Development of Novel Small Molecule Inhibitors by A ...

Development of Novel Small Molecule Inhibitors

of Neurotropic Alphavirus Replication

by

Scott Barraza

A dissertation submitted in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

(Medicinal Chemistry)

in the University of Michigan

2014

Doctoral Committee

Research Professor Scott D. Larsen, Co-Chair

Associate Professor David J. Miller, Co-Chair

Professor John Montgomery

Professor Henry I. Mosberg

ii

Table of Contents

List of Figures .................................................................................................................... iv

List of Schemes .................................................................................................................. vi

List of Tables ..................................................................................................................... ix

List of Abbreviations ......................................................................................................... xi

Introduction ........................................................................................................ 1 Chapter I.

Arboviruses: An Emerging Public Health Threat ........................................................... 1

Neurotropic Alphavirus Virus Biology ........................................................................... 4

Project History ................................................................................................................ 6

Reduction of Polar Surface Area ...................................................................... 9 Chapter II.

Rationale ......................................................................................................................... 9

SAR ............................................................................................................................... 12

Synthesis ....................................................................................................................... 20

Conclusion .................................................................................................................... 24

Reduction of Molecular Weight .................................................................... 26 Chapter III.

Rationale ....................................................................................................................... 26

iii

SAR ............................................................................................................................... 29

Synthesis ....................................................................................................................... 49

Conclusion .................................................................................................................... 59

Conformational Restriction ........................................................................... 60 Chapter IV.

Rationale ....................................................................................................................... 60

SAR ............................................................................................................................... 63

Synthesis ....................................................................................................................... 73

Conclusion .................................................................................................................... 88

Metabolic Stability ......................................................................................... 89 Chapter V.

Rationale ....................................................................................................................... 89

SAR ............................................................................................................................... 90

Synthesis ....................................................................................................................... 98

Conclusion .................................................................................................................. 100

Future Directions ......................................................................................... 101 Chapter VI.

Experimental Data ..................................................................................... 104 Chapter VII.

Bibliography ................................................................................................................... 233

iv

List of Figures

Figure 1. Equine Encephalitis Virus Maintenance Cycle. .................................................. 3

Figure 2. Alphavirus Single-Cell Reproductive Cycle. ...................................................... 5

Figure 3. Natural WEEV Genome and WEEV Replicon. .................................................. 6

Figure 4. HTS Discovery Hit CCG-32091 and Initial Development.................................. 8

Figure 5. Polar Surface Area Contribution per Heteroatom. ............................................ 11

Figure 6. Lead Compounds. .............................................................................................. 12

Figure 7. Right-Hand Chain Length-Dependence. ........................................................... 18

Figure 8. Development of Lead CCG-205432. ................................................................. 26

Figure 9. Sites for Molecular Weight Reduction. ............................................................. 29

Figure 10. WEEV Antiviral Activity for CCG-205432 and CCG-206381. ..................... 34

Figure 11. In Vivo Pharmacokinetic Data for CCG-205432 and CCG-206381. .............. 35

Figure 12. Conformational Restriction Plan for Arene Analogs. ..................................... 42

Figure 13. Spectrum Antiviral Data for Selected Analogues. .......................................... 46

v

Figure 14. Effect of Selected Analogues on Transfected Gene Expression in BSR-T7

Cells. ................................................................................................................................. 48

Figure 15. Steric Approach to Conformational Restriction. ............................................. 61

Figure 16. Tethering Approach to Conformational Restriction. ....................................... 62

Figure 17. Ring-Locking Approach to Conformational Restriction. ................................ 62

Figure 18. Activity of Various Analogs Against Live Virus. ........................................... 69

Figure 19. Predicted Sites of Metabolism in CCG-205432, Rank Ordered...................... 90

vi

List of Schemes

Scheme 1. General Preparation of Low TPSA Analogs. .................................................. 20

Scheme 2. Preparation of CCG-204055 and CCG-205476. ............................................. 21



Scheme 5. Preparation of CCG-211826............................................................................ 23

Scheme 6. General Preparation of Secondary Amine Analogs. ....................................... 24

Scheme 7. Preparation of Pyrrole and Imidazole Analogs. .............................................. 49

Scheme 8. Preparation of Azetidine Analogs. .................................................................. 50

Scheme 9. Preparation of Acyclic Amide and Urea Analogs. .......................................... 51

Scheme 10. Preparation of the Right-Hand Amine Dihydrochloride 36. ......................... 52

Scheme 11. Preparation of N-Benzyl and N-Benzoyl Pyrrolidine Analogs. .................... 52

Scheme 12. Preparation of Anthranilamide and Salicylamide Analogues. ...................... 54

Scheme 13. Preparation of Benzyl Anthranilamide Analogues........................................ 55

Scheme 14. Preparation of Phenethyl Anthranilamide Analogue CCG-222983. ............. 55

vii

Scheme 15. Preparation of Benzyl and Benzoyl Benzoate Analogues. ............................ 56

Scheme 16. Preparation of Carbazole Analogues. ............................................................ 57

Scheme 17. Preparation of Quinolone Analogues. ........................................................... 58

Scheme 18. General Preparation of Rigid Benzamide Analogs. ...................................... 74

Scheme 19. Preparation of 2-(aminomethyl)indane 75. ................................................... 74

Scheme 20. Preparation of Racemic 2-(2-Aminoethyl)indane. ........................................ 75

Scheme 21. Preparation of 7-Methylindole CCG-206447. ............................................... 76

Scheme 22. Preparation of 3-Methylindole CCG-205431. ............................................... 76

Scheme 23. Preparation of pyrrolidopiperidine Analogs CCG-206485 and CCG-212394.

........................................................................................................................................... 77

Scheme 24. Preparation of Inverse Amide CCG-212052. ................................................ 78

Scheme 25. Preparation of Shifted Amide CCG-224001. ................................................ 78

Scheme 26. Preparation of (E)- and (Z)-alkene Analogs. ................................................. 80

Scheme 27. Preparation of Urea Analog CCG-212390. ................................................... 81

Scheme 28. Preparation of Tetrahydropyridine Analog CCG-211823. ............................ 82

Scheme 29. Preparation of Methylpiperidine Analog CCG-211829. ............................... 83

viii

Scheme 30. Preparation of Exo Bicyclic Analog CCG-222980. ...................................... 84

Scheme 31. Preparation of Bridged Analog CCG-224000. .............................................. 84

Scheme 32. Preparation of the Spiro Analog CCG-224220. ............................................ 85

Scheme 33. Preparation of Azetidine-Containing CCG-224002. ..................................... 86

Scheme 34. Preparation of Azapane Analog CCG-222661. ............................................. 87

Scheme 35. Preparation of 4-Fluoropyrrole Analogs. ...................................................... 98

Scheme 36. Preparation of 6-Azaindole Analog CCG-211751. ....................................... 99

ix

List of Tables

Table 1. WEEV Replicon Data for Alicyclic Low TPSA Analogs. ................................. 13

Table 2. WEEV Replicon Data for Aromatic Low TPSA Analogs. ................................. 15

Table 3. MDR1 Recognition Data for Indole and Thienopyrrole leads. .......................... 16

Table 4. WEEV Replicon Data for Amine Analogs. ........................................................ 19

Table 5. WEEV Replicon and ADME Data for Pyrrole and Imidazole Analogs. ............ 30

Table 6. WEEV Replicon Data for Acyclic Low MW Analogs. ...................................... 36

Table 7. WEEV Replicon Data for Pyrrolidine Analogs. ................................................. 37

Table 8. WEEV Replicon and In Vitro ADME Data for Arene Analogs. ........................ 39

Table 9. WEEV Replicon Date for Rigid Arene Analogs. ............................................... 43

Table 10. Antiviral Data for Select Analogues ................................................................. 45

Table 11. WEEV Replicon Data for Methylindole Analogs. ........................................... 64

Table 12. WEEV Replicon Data for Rigid Benzamide Analogs. ..................................... 65

Table 13. WEEV Replicon Data Comparisons of Analogs Based on CCG-205432 and

CCG-206382. .................................................................................................................... 67

x

Table 14. WEEV Replicon Data for Central Piperidine Analogs. .................................... 71

Table 15. MLM Metabolic Stabilities for Thienopyrrole and Indole Leads. .................... 91

Table 16. MLM Metabolic Stabilities For Indole and Pyrrole Analogs. .......................... 93

Table 17. MLM Stability and WEEV Replicon Data for Select Analogs. ....................... 94

Table 18. MLM Stability and WEEV Replicon Data for Select Analogs. ....................... 95

Table 19. WEEV Replicon Data for Fluorinated Analogs. .............................................. 97

xi

List of Abbreviations

ADME Absorption, distribution, metabolism, excretion

BBB Blood-brain barrier

BSL Bio-safety level

CC50 Cytotoxic concentration, half maximal

CEV California encephalitis virus

CNMR Carbon nuclear magnetic resonance spectroscopy

CNS Central nervous system

CPE Cytopathic effect

CYP Cytochrome P450

DMSO Dimethylsulfoxide

EEV Equine encephalitis virus

EEEV Eastern equine encephalitis virus

EMCV Encephalomyocarditis virus

fLUC Firefly luciferase

xii

FMV Fort Morgan virus

HNMR Proton nuclear magnetic resonance spectroscopy

HPI Hours after infection

HPLC High-performance liquid chromatography

HTS High-throughput screen

IC50 Inhibitory concentration, half maximal

IR Infrared spectroscopy

LC Liquid chromatography

MDCK Madin-Darby canine kidney cells

MDR1 Multidrug resistance protein 1

MLM Mouse liver microsome

MOA Mechanism of action

MOI Multiplicity of infection

MS Mass spectrometry

MS/MS Tandem mass spectrometry

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide

xiii

MW Molecular weight

NMR Nuclear magnetic resonance spectroscopy

PAMPA Parallel artificial membrane permeability assay

PFU Plaque-forming unit

Pgp P-glycoprotein

Rho123 Rhodamine 123

SAR Structure-activity relationship

SEAP Secreted embryonic alkaline phosphatase

SEM Standard error of the mean

SD Standard deviation

SINV Sindbis neuroadapted virus

TPSA Topological polar surface area

VEEV Venezuelan equine encephalitis virus

WEEV Western equine encephalitis virus

1

Introduction Chapter I.

Arboviruses: An Emerging Public Health Threat

New and emerging viruses pose an immense public health risk to the world

population,1, 2

due in large part to a lack of effective vaccines and treatments, although

inadequate public health responses may also play a significant role.3 Mosquito-borne

viruses, collectively called arboviruses (arthropod-borne viruses), constitute a large

portion of this risk because of the ubiquity of mosquitoes and the potential for mosquito-

human transmission.4-6

Increasing urbanization and mosquito range expansion further

complicates the threat of potential arboviral pandemic.4-6

Among emerging New World

arboviruses, the equine encephalitis viruses (EEVs) (Western, Eastern, and Venezuelan)

are of particular interest, and are listed as category B agents of biodefense concern due in

part to their potential use in bioterrorism.7-9

EEVs are neurotropic arboviruses that infect the central nervous system (CNS) of

vertebrates, causing debilitating inflammation and damage to the brain.10-12

They are

members of the genus Alphavirus of the Group IV family Togaviridae,13

of which more

than ten members cause significant disease in humans, and all are arboviruses.1 They are

also RNA viruses, and their emerging status may be attributed to this characteristic,2

largely due to poor genome replication fidelity and high mutation rates common to RNA

viruses.14

In nature, EEV existence is maintained by a cycle between passerine birds and

the mosquitoes that prey exclusively on them (Figure 1).15, 16

Infection in humans and

2

animals occurs when other mosquitoes, the so-called bridging vectors, pass the virus

along from birds.17

The natural infection rate in humans, while underreported, is

nonetheless low: since 1964, only 639 cases of Western equine encephalitis virus

(WEEV)18

and 220 cases of Eastern equine encephalitis virus (EEEV) infection have

been confirmed.19

Still, the clinical manifestations of infection can be severe. For victims

of WEEV, the fatality rates may be as low as 3%,20

but neural sequelae may persist well

after infection.21

Young children exhibit seizures and paralysis,22-24

for example, and

upwards of 60% continue to do so for months or years after the virus has disappeared.25

Adults, too, may experience persistent disease-associated impairment.24

Of greater

concern is EEEV, which boasts a mortality rate in excess of 30-40%, and even as high as

70% among some strains,19

and is also likely to cause permanent neurological damage in

survivors.26-28

On the other hand, Venezuelan equine encephalitis virus (VEEV), while

least likely to cause debilitating symptoms in humans,29-31

may have high agricultural

costs associated with infection as equine mortality rates have been observed to exceed

80-90% during epizootic pandemics. Furthermore, unlike with WEEV and EEEV

infections, horses and humans are not dead-end hosts and may continue to spread the

virus.16

3

Figure 1. Equine Encephalitis Virus Maintenance Cycle.

Passerine birds serve as reservoirs for many of the New World alphaviruses, but all

alphaviruses are transmitted to humans and susceptible animals by arthropods.

After World War II, many of the early developers of biological warfare found that

the equine encephalitis viruses and related alphaviruses were highly amenable to

weaponization.9, 16, 32

As stated from a report by the US Army Medical Research Institute

for Infectious Diseases, “Few [viruses] possess as many of the required characteristics for

strategic or tactical weapon development as the alphaviruses.”16

The reasons are simple:

they can be produced in large quantities, they are stable and many are infectious as

aerosols, many are fatal (or at the very least, incapacitating), they are relatively simple to

engineer (and make more lethal), and defensive vaccine development is hindered by the

existence of multiple serotypes. Many nations have long since shut down their offensive

bioweapon programs (the United States in 1969), but today there is a growing threat from

small groups of bioterrorists.9, 16, 33

There currently exist vaccines for WEEV34

and a few of the other alphaviruses,35,

36 but they require multiple shots, confer little immunoprotection, and are available only

4

to researchers.15, 16, 35

It has also been demonstrated that potential vaccines against one

alphavirus may interfere with vaccination against other alphaviruses.37-39

From a public

health perspective, this underlines the need for an effective antiviral, but there are no

therapeutics available for treating infection once the virus has colonized the CNS.15, 40

Therefore, the focus of our research is the development of new, novel compounds for the

pharmacological intervention of CNS infection by alphaviruses.

Neurotropic Alphavirus Virus Biology

The reproductive life cycle of neurotropic alphaviruses is among the most simple

of viruses, and is largely localized to the cytoplasm of the host cell.13

As shown in Figure

2, the first step (step 1) of reproduction involves binding of the virion to specific host

receptors displayed on the exterior of the cell membrane, and receptor-mediated

endocytosis draws the virion into the cell. Upon acidification of the vesicle, the virion is

uncoated and released into the cellular environment (step 2), where it may release its

simple positive-sense single-stranded RNA ((+)ssRNA) genome (step 3.) The genome

may be directly translated upon cellular entry by cytoplasmic ribosomes to produce

protein and enzyme products that facilitate amplification of the genome (step 4), or it

may be transcribed to produce many copies of the original genome (step 5). The copies

may be involved in further translation, or they may be packaged in the capsid of new

virion particles (steps 5, 9, and 10.) Some copies are involved in the translation of

membrane-bound proteins in the secretory pathway, where they will be involved in

recruitment of capsid proteins and assembly of new virion particles, and will ultimately

5

form the viral envelope (steps 6 through 8). Finally, budding occurs and the mature virion

is released (step 11.)13, 41-45

Figure 2. Alphavirus Single-Cell Reproductive Cycle.

Overview of the alphavirus life cycle. Our research focuses on the development of

inhibitors of replication (step 5).

Mosquitoes transmit EEVs to hosts upon biting, causing a localized infection that

may escalate to observable viremia after several days.46-48

The mechanism of invasion of

the CNS is a contentious subject, with some evidence suggesting that the olfactory nerves

are the major route as opposed to the direct crossing of the blood-brain-barrier (BBB).49-

54 Nevertheless, once the virus is established in the CNS, the recruitment of

immunological cells is responsible for encephalitis, but the majority of serious brain

destruction is caused by the induction of neuronal cell death.43, 55

Other cell types are

6

susceptible as well, but form of death is not uniform: glial cells chiefly undergo

apoptosis, whereas neuronal cells usually die via necrotic pathways.55-59

Project History

High-Throughput Discovery. In search of potential antiviral leads, the David

Miller lab executed a high-throughput screen (HTS) of a >50,000-compound library at

the University of Michigan Center for Chemical Genomics (CCG).60

Central to the screen

was a cell-based assay that utilized WEEV replicon plasmids encoding the firefly

luciferase reporter gene.

Figure 3. Natural WEEV Genome and WEEV Replicon. Structural genes were excised and replaced with the reporter gene fLUC, coding for

firefly luciferase. In this way, the WEEV replicon is incapable of producing infectious

virus and may be used without special precautions.

WEEV Replicon Assay. Inhibitory concentrations (IC50 values) were determined

using a WEEV replicon assay developed by our collaborators in the lab of Prof. David

Miller.60

BSR-T7/C3 cells were transfected with a complementary plasmid (pWR-LUC)

encoding a majority of the WEEV genome (Figure 3); however, most of the structural

genes are replaced with the reporter gene firefly luciferase.61

In this way, the replicon

cannot produce infectious virus and thus requires less stringent biosafety containment

7

measures. BSR cells constitutively express bacteriophage T7 RNA polymerase, for which

promoters and terminators exist in the plasmid to maintain high levels of transcription of

the WEEV replicon. Therefore, control cells will produce a baseline luminescence while

cells treated with active inhibitors will show a marked decrease from baseline, from

which an IC50 can be calculated.

MTT Reduction Assay. Cytotoxic concentrations (CC50 values) were established

using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide

(MTT) assay in BSR cells used in the WEEV replicon assay by the lab of Prof. David

Miller.60, 62

In this classical measure of cellular viability, the tetrazolium compound MTT

is reduced to a dark purple formazan dye by oxidoreductase enzymes in living cells. Cells

stressed or killed by cytotoxic compounds exhibit diminished metabolic activity and, as a

result, are less or not at all capable of reducing the MTT to the colorful dye. Therefore,

CC50 values are calculated from the drop in absorbance relative to a healthy baseline

control.

HTS Triage. Because luciferase served as a marker for disruption of genome

replication, those compounds with the lowest IC50 values in the initial HTS were

submitted to secondary and tertiary validation assays. The secondary assay utilized

replicons containing the SEAP reporter gene to indicate transcriptional activity and

confirm that activity was not an artificial construct of luciferase inhibition,63

and the

tertiary assay screened for compounds with selectivity indices (CC50/IC50) greater than

five. One compound, CCG-32091, possessed favorable potency and selectivity, and its

activity was further verified in cultured human neurons in the presence of the live

alphaviruses SINV (Sindbis virus) and FMV (Fort Morgan Virus), which are surrogates

8

for WEEV that require lower level biosafety containment facilities. Limited structure-

activity relationships (SAR) among commercial thienopyrroles related to CCG-32091

showed promising activities and selectivity ratios, as well as a clearly defined SAR

spanning two orders of magnitude potency,60

so the compound was selected for further

optimization.

Figure 4. HTS Discovery Hit CCG-32091 and Initial Development.

The thienopyrrole was substituted with bioisosteric indole (blue), and the known

metabolism-prone furan was replaced with benzyl functionality (red).

Initial SAR. The initial approach was replacement of the thienopyrrole core of

CCG-32091 with an indole core (Figure 4).64

This was undertaken for several reasons:

indoles are generally more metabolically stable than thienopyrroles,65

are cheaper and

synthetically more robust, and phenyl and thiophene are well established bioisosteres.66, 67

CCG-102516, synthesized by Kyle Bolduc, was developed from this rationale. It

contains an indole core, a more lipophilic chloro-substituted N-benzyl group, and a

benzylamide predicted to be more metabolically stable relative to the furylmethylamide

of CCG-32091.68, 69

This initial strategy was successful, as CCG-102516 proved to be

nearly twice as potent as CCG-32091.

9

Reduction of Polar Surface Area Chapter II.

Rationale

The Blood-Brain Barrier. Neurotropic alphaviruses replicate to high titer within

the CNS,70

necessitating development of CNS-penetrant antiviral agents. This is

especially important because clinical manifestations may appear well after the systemic

virus titer has dropped to immeasurable levels, days after initial transmission.71-73

This

places an enhanced emphasis upon retaining physical properties predictive of both good

pharmacokinetics and CNS penetration while optimizing both in vitro and in vivo

activity. The most challenging barrier to CNS entry is the blood-brain barrier (BBB).74-77

The BBB is structurally distinct from other membrane obstacles; due to diminished

pinocytosis and the presence of tight-junctions, most drugs must cross the BBB via

transcellular passive diffusion alone.74, 77

However, there are a number of common

features among successful CNS-active drugs that enhance passive BBB transit, including

low molecular weight (< 400-450), low topological polar surface area (< 60–70 Å2), and

positive logD (~1-3).74-76, 78, 79

MDR1 Recognition Assays. Transporter-mediated efflux is a major hurdle to a

CNS drug’s biological activity in vivo.80, 81

In its protective role, the blood-brain barrier

(BBB) has a number of efflux transporters to facilitate removal of xenobiotics from the

CNS, of which MDR1 is generally the most important.80, 82

Neurotropic alphaviruses

replicate to high titer in the CNS, thereby necessitating the development of transporter-

10

evasive inhibitors to elicit the greatest antiviral effect. It is therefore important to generate

analogs with little to no affinity for MDR1 early in the drug development process.

Over the course of the project, two recognition assays were employed to evaluate

MDR1 recognition of WEEV inhibitors in the lab of Prof. Richard Keep.83

Initially, our

collaborators utilized a simple experiment that measured the impact of our analogs on the

efflux of tritiated (3H) vinblastine, a known MDR1 substrate, from MDCK cells that

expressed human MDR1 (MDR1-MDCKII cells).83, 84

This specific assay was employed

in early evaluations of WEEV inhibitor MDR1 interactions, such as those reported for

low topological polar surface area (TPSA) analogs in this chapter, but later the

Rhodamine 123 uptake assay was employed (see Chapter III). In the 3

H-vinblastine

uptake assay, if the test compound of interest has no interaction with MDR1, the 3H-

vinblastine concentration inside the cell will drop as the substrate is pumped out. If the

test compound does interact with MDR1, the measured 3H-vinblastine concentration will

be higher within the cell. However, due to the nature of the experiment, it cannot be said

whether or not analogs are simple blockers of MDR1 or actual substrates. Regardless, the

data are useful for identifying compounds with low recognition by MDR1 (and thus

potential for efflux) and was used for the prioritization of compounds for further

development.

Initially, we were most interested in maximizing potential for achieving CNS

penetration by reducing the TPSA of the inhibitors. TPSA is a measure of the surface

area of all polar atoms in a molecule, predominately heteroatoms like oxygen and

nitrogen.79, 85, 86

Additionally, polar substituents possess high desolvation penalties and

reduce passive permeability due to repulsive interactions with highly nonpolar lipid

11

membranes.87, 88

High TPSA is also associated with greater recognition by efflux

transporters.89, 90

The total heteroatom contribution to the TPSA of CCG-102516 is

presented in Figure 5; it is evident that heteroatoms with electrons engaged in resonance

have little impact, whereas heteroatoms with a complement of free electrons contribute

more to the TPSA.

Figure 5. Polar Surface Area Contribution per Heteroatom.

Total TPSA and individual contributions were calculated using ChemDraw’s chemical

properties calculator.

BBB-PAMPA Assay. Due to the importance of BBB penetration to our

compounds, and the role of TPSA in enhancing this property, we estimated the passive

membrane permeability for select compounds using BBB-PAMPA (blood-brain barrier

parallel artificial membrane permeability assay) from pION, Inc.,91, 92

and utilized a

cosolvent system due to the poor aqueous solubility of early compounds.93

The traditional

PAMPA assay was developed as a useful low-cost tool for assessing potential passive

membrane permeability for drug-like compounds in the gut, and was found to correlate

well with higher-cost cellular assays.94, 95

However, the BBB is structurally and

compositionally distinct from other membranes, causing PAMPA to often report false

positives; for this reason, BBB-PAMPA was developed and was found to possess

12

superior correlation with BBB passive permeability.96

It is also an important complement

to the MDR1 recognition assay because the MDR1 assay correlates poorly with BBB

passive permeability – due to the different lipid composition of the cells – but contains

efflux transporters not present in BBB-PAMPA.97

While the TPSA of CCG-102516 is within the range of successful CNS drugs,

which have TPSAs significantly lower than other drug classes,74

it is on the higher end of

that range, and we felt it was important to identify low TPSA features that could serve as

new templates throughout other aspects of the SAR campaign in order to maintain

optimal properties predictive of BBB-penetration. Furthermore, it was important to

determine whether TPSA could be reduced with maintenance of anti-viral activity.

SAR

Initial analogs were based on CCG-203880, the equipotent N-4-fluorobenzyl

analog of CCG-102516 (Figure 6) because of its lower molecular weight (see Chapter

III). A general feature of these analogs was the replacement of the right-hand secondary

amide with alternative linkers.

Figure 6. Lead Compounds.

CCG-102516 and its equipotent fluoro analog CCG-203880.

13

Table 1. WEEV Replicon Data for Alicyclic Low TPSA Analogs.

CCG No. X R IC

50

(µM)a

CC50

(µM)b

MDR1

(% vinblastine

uptake)c

TPSAd

203880† F 15 ± 2 >100 1,495 53

203941 F >100 >100 --- 27

205421 F 14 ± 1 24 ± 1 --- 44

205475 F >100 >100 --- 41

203945 F 23 ± 2 >100 --- 44

204055 F 12 ± 1 96 821 27

205476 Cl 10 ± 2 38 --- 27

aInhibition of luciferase expression in WEEV replicon assay. bCell viability determined

by the MTT reduction assay. Values are mean of at least n=3 independent experiments.

dTPSA was predicted using ChemDraw’s chemical properties calculator. †Synthesized by

Janice Sindac.

Alicyclic Low TPSA SAR. As seen in Table 1, we were able to demonstrate that

TPSA could be reduced substantially in some cases without impacting activity or toxicity

(e.g. alicyclic analogs CCG-203945 and 204055). Aromatic rings are capable of

engaging in numerous types of bonding interactions – like ring-stacking or weak CH

14

hydrogen bonding – not available to alicycles. That the active low TPSA analogs bear

non-aromatic heterocycles and carbocycles suggests that the binding in this region may

be largely dependent on hydrophobic interactions between the ring and lipophilic

residues in the unknown binding site. Also of interest is the difference in activities

between CCG-205421 and CCG-205475. Both compounds are hydrogen bond acceptors,

but only CCG-205421 is also a hydrogen bond donor, perhaps forming a key contact

with an acceptor in the binding site. CCG-205421 is also tetrahedral about the sp3

alcohol carbon, unlike the sp2 carbonyl carbon in CCG-205475. This could indicate that

CCG-205421 has geometry that enhances interactions with unknown binding site

contacts.

Aromatic Low TPSA SAR. As seen in Table 2, like the alicyclic analogs, we

found TPSA could be halved in the aromatic analogs while maintaining favorable

potency and toxicity profiles (e.g. aromatic analogs CCG-203942 and CCG-203945),

placing WEEV inhibitor properties well within the range of successful CNS drugs.

Intriguingly, the SAR of ketone CCG-203943 and alcohol CCG-204056 matched that of

their alicyclic counterparts CCG-205475 and CCG-205421, respectively (Table 1). We

also noted that benzylamine CCG-205420 was substantially more potent than its

benzylether analog CCG-203944, but this result may be an artificial product of its high

toxicity. Curiously, benzyl piperazine analogs (e.g. CCG-203942) were eqipotent with

the lead, in contrast to the inactive cyclohexylmethyl piperazine CCG-203941 (Table 1).

This challenged the assumption that lipophilicity was the most important factor for

binding in this region of molecule.

15

Table 2. WEEV Replicon Data for Aromatic Low TPSA Analogs.

CCG No. X R IC

50

(µM)a

CC50

(µM)b

MDR1

(% vinblastine

uptake)c

TPSAd

203880† F 15 ± 2 >100 1,495 53

203943 F >100 >100 --- 41

204056 F 15 ± 0.4 40 --- 44

204020 F >100 >100 --- 27

205420 F 22 ± 3 39 --- 36

203944 F >100 >100 --- 33

203942 F

12 ± 1 >100 764 27

206372 Cl

12 ± 1 >100 --- 27

211827 Cl

7.2 >100 --- 27

211826 Cl

12.4 >100 --- 39

aInhibition of luciferase expression in WEEV replicon assay.

bCell viability determined


†Synthesized by Janice Sindac. dTPSA was predicted using ChemDraw’s chemical

properties calculator.

16

Table 3. MDR1 Recognition Data for Indole and Thienopyrrole leads.

CCG No. R MDR1 Recognition (%

vinblastine uptake)a

102516†

534.0

203880†

1495.6

203881†

433.3

32088*

694.1

aMDR1 recognition was assessed by measuring uptake

3H-vinblastine at 30 µM of test

anti-viral or vehicle. †Synthesized by Janice Sindac. *Synthesized by Bryan

Yestrepsky.

MDR1 Recognition. It was hoped that the best low TPSA candidates from this

assay may serve as new templates for further development. After our initial survey of the

activity of these analogs was complete, CCG-204055 and CCG-203942 were graduated

to the MDR1/vinblastine assay because they showed promising inhibition and low

cytotoxicity. Both of these compounds were found to possess improved MDR1-efflux

results over CCG-203880 (which may be attributed to their lower TPSA compared to

their amide counterparts), and were therefore considered potential new leads. However,

17

initial trends among other scaffolds suggested that chloro-substituted compounds may be

more MDR1-evasive than their fluoro-substituted counterparts (Table 3), prompting us to

prepare and evaluate chloro-substituted analogs of some of the better fluoro-substituted

compounds (e.g. CCG-205476 and CCG-206372, Table 1 and Table 2, respectively).

These compounds were found to be equipotent to their fluoro-substituted cousins.

Unfortunately, these compounds were never submitted to the MDR1 recognition assay

due to the discovery of the significantly more potent 4-pyridylethylamide inhibitor class,

described briefly below and in detail in Chapter III.

TPSA Analog Length-Activity Dependence. While many of the TPSA analogs

feature low polar surface area with retention of activity, it was apparent that there was

still room for potency optimization. Janice Sindac, working on other aspects of the

WEEV project, had established that the right-hand portion of the template must be of a

specific length, where the intervening alkyl chain between the amide and the aromatic is

optimally 2-carbons long (Figure 7).83

Interestingly, propyl linkers abolished activity

entirely. The low TPSA compounds in Table 1 and Table 2 were significantly truncated,

so longer analogs were investigated. Interestingly, analogs with ethyl and propyl linkers

did not perform significantly better than their shorter counterparts (e.g. CCG-206372

compared to CCG-211827, Table 2), nor were they worse. Even CCG-211826, which

bears a 4-pyridylethyl substituent crucial to the activity of later analogs (see Chapter

III), merely maintained activity. This suggested that either important structural features

were not present among the low TPSA analogs or that they were not binding in the same

way as the amide leads. Indeed, later work established the importance of the right-hand

18

secondary amide to activity (see Chapter IV) which is lacking in the low TPSA inhibitor

class.

Figure 7. Right-Hand Chain Length-Dependence.

Chain-length dependence of the right-hand region “R” was found to decrease in activity

such that n=2>1>>3, where n is an intervening number of methylene units.

Secondary Amine Analog SAR. The low TPSA analogs are highly lipophilic.

While this aids in BBB permeation, it decreases water solubility such that aqueous

delivery of compound is impeded. Interestingly, analogs containing ionizable

functionalities, such as amines, exhibited the greatest activity (e.g. piperazine-containing

analogs CCG-203942 and CCG-204055), perhaps because of improved aqueous

solubility. Alternatively, the analogs may simply possess improved permeability, as very

lipophilic compounds may be retained by the membrane, inhibiting their ability to

penetrate the cell. In an effort to exploit this trend, a new generation of right-hand amine-

bearing analogs was prepared (Table 4), but these proved to be quite toxic, especially

those containing secondary amines (e.g. CCG-206486 and CCG-206500), perhaps via

detergent activity (disruption of cellular membranes). On the other hand, tertiary amines

(e.g. CCG-206502 and CCG-206503) were better tolerated but displayed markedly

decreased activity. All of the analogs possessed lower CC50 values than those seen in

19

their amide counterparts, and there was no significant activity improvement. For these

reasons, the amine-containing series was discontinued early. This did not undermine

development efforts of the low TPSA WEEV inhibitor class; overall, we demonstrated

that activity could be maintained with reduced TPSA and – potentially – increased

hydrophilicity.

Table 4. WEEV Replicon Data for Amine Analogs.

CCG No. R IC50 (µM)a CC50 (µM)

b

203880†

14.8 ± 2.1 >100

206486

6.1 ± 0.7 9

206499

>100 >100

206500

6.5 ± 0.3 12

206501

8.9 ± 0.4 16

206502

40.6 ± 0.5 96

206503

25.1 ± 1.9 85


bCell

viability determined by the MTT reduction assay. Values are mean

of at least n=3 independent experiments. †Synthesized by Janice

Sindac.

20

Synthesis

Low TPSA analogs were generally prepared through the alkylation of ethyl 2-

indolecarboxylate 1 with either 4-chloro- or fluorobenzyl chloride 2a or 2b. The N-

alkylated ethyl ester intermediate 3a,b was then saponified to the corresponding

carboxylic acid 4a,b and coupled with the desired amine to give the general structure

5a,b (Scheme 1).

Scheme 1. General Preparation of Low TPSA Analogs.a

aReagents and conditions: (a) K2CO3, DMF, 60 °C, 20 h, 70%; (b) 7 M aq. NaOH, EtOH,

50 °C, 3 h, 97%; (c) HNR1R2, EDC, HOBt, DIPEA, DMF, rt, ~ 24 h.

Many amines R1R2NH had to be prepared due to commercial unavailability. The

1-(piperidin-4-ylmethyl)piperidine portion of the analogs CCG-204055 and CCG-

205476 was first accessed through the Boc-protection and Swern oxidation of

piperidinyl-4-methanol 6. This provided the aldehyde 8 that underwent reductive

amination with piperidine to give the boc-protected amine 9, and this was subsequently

21

deprotected to reveal the amine as a trifluoroacetate salt and coupled with the

appropriately halogenated indole carboxylic acid to yield the desired analogs (Scheme 2).

Scheme 2. Preparation of CCG-204055 and CCG-205476.a

aReagents and conditions: (a) Boc2O, Na2CO3, H2O, THF, reflux, 2 h, 87%; (b) DMSO,

(COCl)2, -78 °C, 30 min; then TEA, -78 °C→0 °C, 3 h, 89%; (c) piperidine, NaCNBH3,

TFE, 3 Å MS, RT, 20 h, 72%; (d) TFA, DCM, RT; (e) 4a or 4b, EDCHCl, HOBT,

DIPEA, DMF, RT, 15-24 h, 54-63%.

The alicyclic analogs CCG-205421 and CCG-205475 were accessed through a

single linear route in which the latter was a direct synthetic derivative of the former

(Scheme 3). In this fashion, the aldehyde 8 was treated with cyclohexylmagnesium

bromide and the resulting Grignard reaction adduct 10 was deprotected to reveal the

secondary amine, which was then coupled with the indole carboxylic acid to afford the

first analog, hydroxyl-containing CCG-205421, which was contaminated with ester from

competing O-acylation. The desired amide was separated from the ester side-product and

confirmed by IR spectroscopy. CCG-205475 was then prepared by simply oxidizing the

alcohol to the ketone under Swern conditions.

22


aReagents and conditions: (a) cyclohexyl-MgBr, Et2O, RT, 2 h, 85%; (b) TFA, DCM,

RT, 15 h; (c) 1-(4-fluorobenzyl)-1H-indole-2-carboxylic acid, EDCHCl, HOBT, DIPEA,

DMF, rt, 24 h, 60%; (d) (COCl)2, DMSO, DCM, -78 °C, 30 min; then TEA, -78 °→RT,

30 min, 58%.


aReagents and conditions: (a) BnBr, Na2CO3, H2O, DCM, reflux, 3 h, 87%; (b) TFA,

DCM, RT, 18 h; ; (c) 1-(4-fluorobenzyl)-1H-indole-2-carboxylic acid, EDCHCl, HOBT,

DIPEA, DMF, rt, 30 h, 52%; (d) Pd(OH)2, H2, HCl, EtOH, THF, RT, 1 h, 77%.

23

The benzylated amine analogs CCG-204020 and CCG-205420 were prepared in

a synthetically distinct, albeit similarly linear fashion, to the previous two compounds

(Scheme 4). The protected 4-aminopiperidine 12 was doubly benzylated with benzyl

bromide and deprotected with TFA to afford the amine salt 14. Coupling with the indole

carboxlic acid 4b gave CCG-204020, and time-sensitive selective mono-debenzylation

provided the singly substituted benzyl amine CCG-205420.

The synthesis of piperazine analog CCG-211826 required prior preparation of a

suitable alkylation partner (Scheme 5) by displacement of alcohol 15 under forcing

conditions with refluxing hydrobromic acid. 1-Boc-piperazine was then alkylated with

the resulting bromo compound 16, deprotected under acidic conditions, and coupled with

the indole carboxylic acid 4a to provide the desired final analog.

Scheme 5. Preparation of CCG-211826.a

aReagents and conditions: (a) conc. HBr, 130 °C, 2 h, 40%; (b) t-butyl piperazine-1-

carboxylate, NaH, DMF, RT, 19 h, 66%; (c) HCl, 1,4-dioxane, RT, 15 min, 100%; (d) 1-

(4-Chlorobenzyl)-1H-indole-2-carboxylic acid, TEA, EDCHCl, HOBT, DCM, RT, 14 h,

63%.

24

Secondary amine analogs were generally prepared through the coupling of the

indole carboxylic acid 4b and piperidine-4-methanol 19 (Scheme 6). The resulting

alcohol adduct was then oxidized under Swern conditions to the aldehyde 20, which

could undergo reductive amination with various amines to provide the desired diversity

of structural type 21.

Scheme 6. General Preparation of Secondary Amine Analogs.

aReagents and conditions: (a) EDCHCl, HOBT, DIPEA, DMF, 94%; (b) DMSO,

(COCl)2, DCM, -78 °C; then TEA, -78 °C→0 °C, 66%; (c) NHR1R2, Na(CN)BH3,

AcOH, THF, EtOH.

Conclusion

The primary goal of this chapter was reduction of topological polar surface area. To

this end, we succeeded in halving the TPSA while maintaining potency, bringing our

compounds well within the optimal range of successful CNS drugs. This work also

identified useful structural modifications (e.g. piperidine-to-piperazine substitutions) and

25

underlined the need to avoid secondary amine functionality due to cytotoxicity issues.

Notably, reduction of TPSA appeared to enhance MDR1 evasion, an important feature

for CNS-active drugs.

26

Reduction of Molecular Weight Chapter III.

Rationale

Janice Sindac, working on other aspects of the WEEV project, had identified

CCG-205432 as a viable new lead due to its substantially improved potency and

solubility over the earlier lead CCG-102516 (Figure 8).83

However, it was clear that

some properties of CCG-205432 required optimization due to its increased molecular

weight (MW) and TPSA, features associated with diminished passive permeability and

thus reduced activity. Due to the rather flat SAR associated with the low TPSA analogs

(see Chapter II), we elected to focus on molecular weight reduction as an alternative

course of action for correcting the physicochemical properties of CCG-205432.

Figure 8. Development of Lead CCG-205432.

Molecular weight (MW) is a property of extreme importance for CNS-acting

drugs, which tend to be of low molecular weight (<450).74, 98

This is crucial, as reduction

27

of molecular weight promotes passive permeability into membranes non-linearly;99

even

a small change in molecular weight can have a large impact on membrane penetration.

Furthermore, reduced molecular weight is associated with lower recognition by efflux

transporters like MDR1,79, 80, 100, 101

an attribute we considered essential for our WEEV

inhibitors.

MDR1 Rho123 Uptake Assay. Our collaborators in the lab of Prof. Richard

Keep discovered that the 3H-vinblastine uptake assay suffered from poor reproducibility

due to complications in acquiring the specific MDR1-MDCKII cell line used in previous

work (see Chapter II). Fortunately, they found that the Rhodamine 123 (Rho123) uptake

assay102

was a superior substitute to the 3H-vinblastine uptake assay, and this was utilized

for the reduced molecular weight analog class. Not only did the Rho123 assay feature

improved reproducibility, but Rho123 is a simple dye whose concentration may be

measured by absorbance and avoids those complications associated with radioactivity.

Both of these assays operate in the same manner; if the test compound of interest has no

interaction with MDR1, the 3H-vinblastine or Rho123 concentration inside the cell will

drop as the substrate is pumped out. If the test compound does interact with MDR1, the

measured 3H-vinblastine or Rho123 concentration will be higher within the cell. Results

for selected analogs are reported as percent effect on Rho123 intracellular concentration

relative to tariquidar, where lower values indicate lesser interaction with MDR1 and

therefore less potential for efflux. Moreover, the Rho123 assay was controlled by

comparing WEEV inhibitors to the efflux of the known MDR1 inhibitor tariquidar.

We were also interested in undertaking MDR1 SAR studies in order to define an

MDR1-recognition pharmacophore, but such work is not always trivial.89

MDR1 binds

28

many different substrates, and traditional SAR, which focuses on biomolecules that bind

specific substrates, is not always up to the task. Further complications arise when MDR1

binds only a few compounds from a class of analogs. There are very general SAR trends

associated with the transporter, but most individual chemotypes must typically be

optimized on a case-by-case basis.

Mouse Liver Microsome Metabolic Stability Assay. Metabolic stability is one

of the most crucial aspects of early drug development, and instability is a potential killer

of lead series.81

It is therefore necessary to improve the stability of a compound to

maximize its potential for in vivo efficacy, especially as half-life is related to a

compound’s biological availability. (See Chapter V for more information regarding

metabolic stability in drug development.) To this end, the half-lives of compounds were

determined by incubating in the presence of Balb/C mouse liver microsomes containing

cytochrome p450 (CYP) enzymes and monitoring the decrease in parent compound

concentration over time. Compounds more susceptible to CYP-mediated metabolism will

decrease more rapidly than those that are more stable. Concentration was measured using

an LC/MS/MS method developed specifically to maximize throughput, and all values

were controlled against a known concentration of a structurally-related internal standard.

The assay and analytical protocols were developed in our lab specifically to fit the needs

of the WEEV project, and details appear in the experimental section.

Kinetic Solubility Assay. The kinetic solubility is a measure of the metastable

solution equilibrium of a compound (pre-dissolved in a solvent like DMSO and added to

aqueous media) that begins to precipitate as its concentration exceeds its ability to

dissolve.79, 103

This can be observed by measuring the absorbance of the sample. The

29

concentration at which the compound precipitates is reported as the kinetic solubility, and

is a useful measure of aqueous solubility that allows for prioritization of compounds of

interest.

SAR

The average CNS drug has a molecular weight of about 319, but the lead

compounds CCG-102516 and CCG-205432 (Figure 8) were much higher than that. We

were therefore interested in exploring the indole and piperidine cores as potential sites for

molecular weight reduction (Figure 9), largely due to the lack of attention these regions

had received until this point. A number of analogs featuring monocyclic heterocycles

lacking the fused phenyl ring of indole, such as pyrroles and imidazoles, were prepared

(Table 5). Azetidine-containing analogs of the piperidine core were also synthesized and

evaluated.

Figure 9. Sites for Molecular Weight Reduction.

30

Tab

le 5

. W

EE

V R

epli

con

an

d A

DM

E D

ata

for

Pyrr

ole

an

d I

mid

azo

le A

nalo

gs.

MW

486

501

437

451

437

ML

M

T1

/2

(min

)e

7.3

± 3

.9

11.5

---

2.9

---

MD

R1

Rec

ognit

i

on (

%

Rho123

upta

ke)

d

---

30.9

± 1

.0

---

-1.2

± 0

.6

---

Sol

(µM

)c

---

31

-63

---

125

-250

---

BB

B-

PA

MP

A

(logP

eff)

-3.6

±

0.3

-4.1

8 ±

0.0

6

---

-5.0

1 ±

0.0

3

---

CC

50

(µM

)b

>100

65.3

>100

>100

>100

IC5

0

(µM

)a

15.6

± 1

.8

0.5

3 ±

0.0

1

77 ±

23

0.6

8 ±

0.0

4

>100

R

---

---

X

---

---

C

C

N

CC

G N

o.

102516†

205432†

204054

206381

206586

31

Tab

le 5

. C

on

tin

ued

.

MW

437

451

437

a Inhib

itio

n o

f lu

cife

rase

expre

ssio

n i

n W

EE

V r

epli

con a

ssay

. bC

ell

via

bil

ity d

eter

min

ed b

y t

he

MT

T r

educt

ion a

ssay

. V

alues

are

mea

n o

f at

lea

st n

=3 i

ndep

enden

t ex

per

imen

ts. c

Log o

f ef

fect

ive

per

mea

bil

ity (

cm/s

) det

erm

ined

usi

ng P

AM

PA

Explo

rer

(pIO

N)

wit

h B

BB

lip

id m

ixtu

re m

easu

red a

t pH

= 7

.4.

dK

inet

ic s

olu

bil

ity m

easu

red u

sing t

he

sam

e as

say m

edia

as

WE

EV

repli

con a

ssay

, ex

cept

wit

h 1

0%

fet

al b

ovin

e se

rum

. e R

hodam

ine

123 u

pta

ke

was

mea

sure

d i

n M

DR

1-M

DC

KII

cel

ls u

tili

zing

Glo

max

Mult

i D

etec

tion S

yst

em (

Pro

meg

a). ‘M

DR

1 r

ecognit

ion’

was

ass

esse

d b

y m

easu

ring u

pta

ke

of

Rhodam

ine

123 i

n t

he

pre

sence

of

MD

R i

nhib

itor,

tar

iquid

ar (

5 µ

M),

30 µ

M o

f te

st a

nti

-vir

al o

r v

ehic

le, an

d c

alcu

lati

ng:

(Cav

– C

veh

)*100/(

Cta

r-C

veh

),

wher

e C

av =

conce

ntr

atio

n o

f rh

odam

ine

123 i

n t

he

pre

sen

ce o

f an

ti-v

iral

, C

veh

= c

once

ntr

atio

n i

n t

he

pre

sen

ce o

f v

ehic

le, C

tar =

conce

ntr

atio

n o

f rh

odam

ine

123 i

n t

he

pre

sen

ce o

f ta

riquid

ar.

In t

he

pre

sen

ce o

f ta

riquid

ar, rh

od

amin

e 123 u

pta

ke

was

1123 ±

54%

of

veh

icle

contr

ols

(n=

44).

f Hal

f-li

fe i

n B

AL

B/c

mouse

liv

er m

icro

som

e in

cubat

ion. V

alues

are

hal

f-li

ves

cal

cula

ted f

rom

the

equat

ion T

1/2

=ln

(2)/

slope,

wher

e th

e sl

ope

was

ln(%

det

ecte

d)

agai

nst

tim

e, p

lott

ed f

rom

the

mea

n o

f at

lea

st n

=3

indep

enden

t ex

per

imen

ts. †

Synth

esiz

ed b

y J

anic

e S

indac

.

ML

M

T1

/2

(min

)f

---

---

---

MD

R1

Rec

ognit

ion

(% R

ho123

upta

ke)

e

0.1

3 ±

0.3

7

---

---

Sol

(µM

)d

>500

---

---

BB

B-

PA

MP

A

(logP

eff)

c

-5.0

6 ±

0.0

4

---

---

CC

50

(µM

)b

>100

>100

>100

IC5

0

(µM

)a

>100

>100

22 ±

8

R

X

N

C

C

CC

G N

o.

208916

208829

208915

32

Heterocyclic Monocycle SAR. The imidazole analogs (CCG-206586 and CCG-

208916) were completely inactive, presumably due to the enhanced ionizability and

diminished lipophilicity arising from the presence of two azine nitrogens (Table 5).

These properties would be expected to impede passive membrane permeability.

Gratifyingly, the pyrrole analog CCG-206381 was nearly equipotent with indole CCG-

205432, and boasted improved aqueous solubility and a 50 Da improvement in molecular

weight (Table 5). Attempts to reduce molecular weight of CCG-206381 further, by

replacing the central piperidine with azetidine, returned significantly less active

compounds (Table 5). This may be attributed to the length-activity dependence discussed

in Chapter II, as the small 4-membered ring is much smaller and shorter than the larger

6-membered piperidine. Curiously, the pyrrole analog of CCG-102516 (CCG-204054)

was substantially less active than its lead.

Potential for BBB Penetration. Passive permeability is an excellent predictor of

successful BBB penetration, and high permeability may also overcome the effect of

efflux transporters.79

Therefore, compounds with good BBB permeability are of central

importance to our development of CNS active inhibitors, and the pyrrole and imidazole

analogs were evaluated for their potential to permeate the BBB. The notion that the

significantly diminished activity of the imidazole analogs was related to increased

ionizability and lipophobicity was supported by the high aqueous solubility and low

BBB-PAMPA permeability of CCG-208916 seen in Table 5 (compounds with log Peff >

4.7 are considered to be highly permeable, whereas those with log Peff 6 are

considered poorly permeable). CCG-206381, as expected, possessed improved aqueous

solubility (Table 5), but appeared to have reduced permeability as measured by the BBB-

33

PAMPA assay. It is important to note that PAMPA assays are not perfect models of

membranes because of reduced surface area and the presence of multiple (>100) lipid

layers. Therefore, it could not be confidently concluded that CCG-206381 was in fact

less permeable than CCG-205432.

Recognition by efflux transporters is highly correlated with diminished BBB

permeation, so selected analogs were tested for recognition by MDR1 in the Rho123

assay, using the known MDR1 substrate tariquidar as a control (Table 5). Significantly,

CCG-205432 interacted with MDR1 to an extent equal to 30% that of tariquidar, whereas

CCG-206381 had virtually no detectable interaction with the transporter. This indicated

that CCG-206381 could potentially evade MDR1 and thus achieve a higher in vivo brain

concentration than CCG-205432.

Antiviral Activity. Due to its improved aqueous solubility and reduced molecular

weight, CCG-206381 was advanced into live virus studies and compared to CCG-

205432. In these assays, cells were infected with live WEEV and the number of plaques –

often visually distinct zones caused by infection – were counted and used to extrapolate

viral concentration after treatment with varying concentrations of test compound.

Viability was determined by measuring the number of living cells with the MTT assay.

CCG-205432 reduced viral titer by over two log units, and CCG-206381 was able to

successfully reduce viral titer by about one log unit (Figure 10A). Both compounds also

comparably conferred protection to infected cells (Figure 10B). Together, these results

indicated that, while only modestly less active, pyrrole could serve as a viable reduced

molecular weight alternative to indole.

34

Figure 10. WEEV Antiviral Activity for CCG-205432 and CCG-206381.

(A) HEK293 cells were infected with WEEV at a multiplicity of infection (MOI) of 1 and

simultaneously treated with decreasing concentrations of the indicated compound. Virus

titers in cell culture supernatants were determined at 24 hpi by plaque assay. Calculated

IC50 values for CCG-205432 and CCG-206381 were 1.8 ± 0.1 and 13.9 ± 6.9,

respectively. (B) Assay utilized the alphavirus WEEV. Infections were done in cultured

human BE(2)-C neuronal cells. Viability was measure using an MTT assay, and viral

titers were measured using a plaque assay. Values are mean ± SEM of n = 3-4

independent experiments. P value <0.05* or 0.005

** compared to DMSO control.

In Vivo Pharmacokinetic Properties. Despite its reduced molecular weight,

CCG-206381 was less permeable in the BBB-PAMPA assay (Table 5) than its indole

analog CCG-205432. However, while lead CCG-205432 could not achieve a measurable

brain concentration in mouse pharmacokinetic studies, CCG-206381 did register brain

permeation at the early time points (Figure 11). This confirmed our prediction that

reduction of molecular weight would enhance BBB permeability; due to significantly

reduced MDR1 recognition, we can credit CCG-206381’s BBB penetration with

enhanced evasion of efflux transporters despite its worse BBB-PAMPA permeability.

35

However, the instability of CCG-206381 towards CYP-mediated metabolism compared

to CCG-205432 in the mouse liver microsomal (MLM) assay (see Chapter V for more

information) was reflected in its pharmacokinetic profile (Figure 11). CCG-205432 had

an MLM half-life of 11.5 minutes and was measurable in rat plasma for over 20 hours,

whereas CCG-206381 had an MLM half-life of 2.9 minutes and was measurable in rat

plasma or brain samples only up to one hour. Overall, this data indicated that reduction of

molecular weight was a viable tactic in achieving brain penetration and, therefore, in vivo

efficacy.

Figure 11. In Vivo Pharmacokinetic Data for CCG-205432 and CCG-206381.

CCG-205432 and CCG-206381 possess different pharmacokinetic profiles. Balb/C mice

were dosed and sacked at various time points (David Irani lab). Compound

concentrations in plasma and brain samples were evaluated via LC/MS/MS by the lab of

Prof. Duxin Sun.

0

20

40

60

80

100

120

0 10 20

Con

cen

trati

on

(n

g/m

L)

Time (hours)

CCG-205432 Plasma/Brain

Concentration-Time

Plasma

Brain

0

20

40

60

80

100

120

0 10 20

Con

cen

trati

on

(n

g/m

L)

Time (hours)

CCG-206381 Plasma/Brain

Concentration-Time

Plasma

Brain

36

Table 6. WEEV Replicon Data for Acyclic Low MW Analogs.

No. R IC50

(µM)a

CC50

(µM)b MW

206381 --- 0.68 ± 0.04 >100 451

208825

60.2 ± 4.6 >100 401

208827

40.9 ± 5 >100 415

208848

>100 >100 386

208846

>100 >100 400


bCell

viability determined by the MTT reduction assay. Values are mean of

at least n=3 independent experiments.

Acyclic Analog SAR. Having established that a smaller scaffold than indole

could retain antiviral activity and improve BBB permeability, a variety of additional

analogs was synthesized (Table 6) that featured complete excision of the left-hand

heterocycle. Many of these analogs possess molecular weights in the low 400 to high 300

g/mol range, bringing these values substantially closer to successful CNS drugs than any

WEEV inhibitor synthesized to this point. However, while the pyrrole analogs indicated

that the indole left-hand core was not a necessary component of the SAR, it was

immediately evident that complete removal of the heterocycle eliminated activity. Only

ureas CCG-208825 and CCG-208827 retained any activity in the WEEV replicon assay,

and those were greatly diminished relative to pyrrole CCG-206381. Amides 208848 and

37

208846 were completely inactive. These results suggested that some degree of rigidity is

likely necessary to retain good antiviral activity.

Table 7. WEEV Replicon Data for Pyrrolidine Analogs.

No. X IC50

(µM)a

CC50

(µM)b

MW

206381 --- 0.68 ± 0.04 >100 451

(S)-211758 CH2 13.5 ± 2 91.6 455

(R)-211757 CH2 53.5 ± 12 >100 455

(S)-211754 CO >100 >100 467

(R)-211756 CO >100 >100 467 aInhibition of luciferase expression in WEEV replicon assay.

bCell

viability determined by the MTT reduction assay. Values are mean

of at least n=3 independent experiments.

Pyrrolidine Analog SAR. We therefore turned our attention to cyclic, but non-

aromatic, pyrrolidine analogs (Table 7). Our rationale was that the saturated heterocycles

would possess greater stability towards oxidation than pyrrole, yet provide the rigidity

that was apparently required. While significantly less active than pyrrole CCG-206381,

N-benzyl pyrrolidines CCG-211758 and CCG-211757 still retain some activity. Worth

noting is the small but significant chiral preference for the S-enantiomer, which is four

times more potent than the R-enantiomer. This was consistent with our previous

38

observation that the unknown molecular target for this series of antiviral compounds

possesses some degree of enantiospecificity.64, 83

N-benzoyl pyrrolidines CCG-211754

and CCG-211756 by contrast were completely inactive. This could suggest a need for a

basic nitrogen in the linker, but more likely reflects an unfavorable conformational bias

induced by the planar nature of the amide.

Arene Monocycle SAR. We then synthesized and evaluated a series of arene

derivatives (Table 8) to more closely mimic the pyrrole scaffold but with greater

predicted stability to oxidative metabolism. Several of these compounds possessed low

micromolar activity in the replicon assay, including salicylamide CCG-212392 and

anthranilamide CCG-211824. We anticipated that the aniline moiety might be the most

suitable bioisosteric replacement for pyrrole based on its electronic similarity, and indeed

CCG-211824 was only two-fold less potent than CCG-206381. The benzyl aniline

homologue CCG-222660 proved to be equipotent with the lead (compound CCG-

206381), while further homologation of the anthranilamide (analog CCG-222983)

resulted in a diminishment of activity. This observation of an optimal length is consistent

with what we observed while developing CCG-20543283

and suggests either a well-

defined limit to the size of the unknown binding site, or simply reflects an excessive loss

of entropy needed for binding a long acyclic substituent. Removal of the chloride

(intended to improve metabolic stability by reducing lipophilicity) resulted in compounds

(CCG-222981 and CCG-222982) with greatly reduced potency. Replacement of the

aniline nitrogen of CCG-211824 with oxygen (ether analog CCG-212392) resulted in a

modest loss of activity, while replacement with methylene (benzyl analog CCG-222821)

provided an equipotent analogue.

39

Tab

le 8

. W

EE

V R

epli

con

an

d I

n V

itro

AD

ME

Data

for

Are

ne

An

alo

gs.

MW

501

451

464

463

429

Sol

(µg/m

L)f

31-6

3

125-2

50

>250

ML

M

T1

/2

(min

)e

9

3

18

19

---

MD

R1

Rec

ognit

ion

(% R

ho

123

upta

ke)

d

30.9

± 1

.0

-1.2

± 0

.6

9.3

± 3

.2

47.7

± 7

.2

10.9

± 1

.7

BB

B-

PA

MP

A (

log

Pef

f)c

-4.1

8 ±

0.0

6

-5.0

1 ±

0.0

3

-4.6

8 ±

0.0

9

-4.4

5 ±

0.1

1

-4.7

5 ±

0.0

2

CC

50

(µM

)b

49.9

>100

56.0

>100

>100

IC5

0

(µM

)a

0.5

3 ±

0.0

8

0.6

8 ±

0.0

4

6.1

± 0

.8

1.6

± 0

.2

28.8

± 5

.2

G

---

---

No.

205432

206381

212392

211824

222981

40

Tab

le 8

. C

on

tin

ued

.

MW

477

477

443

491

476

462

Sol

(µg/m

L

)f

>250

>250

>250

ML

M

T1

/2

(min

)e

---

15

---

---

--- 3

MD

R1

Rec

ognit

ion

(% R

ho123

upta

ke)

d

3.9

± 1

.5

59.6

± 7

.5

2.5

± 0

.3

30.7

± 4

.7

1.8

± 1

.3

22.6

± 2

.7

BB

B-

PA

MP

A (

log

Pef

f)c

-4.7

1 ±

0.0

9

-4.3

9 ±

0.0

5

-4.9

3 ±

0.0

3

-4.4

9 ±

0.3

5

-5.0

8 ±

0.0

2

-4.4

0 ±

0.0

6

CC

50

(µM

)b

>100

75.2

92.2

86.0

>50

70.7

IC5

0

(µM

)a

15.4

± 2

.8

0.5

6 ±

0.0

8

19.0

± 1

.7

3.4

9 ±

0.1

>50

1.4

6 ±

0.0

9

G

No.

212393

222660

222982

222983

212391

222821

41

Tab

le 8

. C

on

tin

ued

.

a Inhib

itio

n o

f lu

cife

rase

expre

ssio

n i

n W

EE

V r

epli

con a

ssay

. bC

ell

via

bil

ity d

eter

min

ed b

y t

he

MT

T r

educt

ion a

ssay

. V

alues

are

mea

n o

f at

lea

st n

=3 i

ndep

enden

t ex

per

imen

ts. c

Log o

f ef

fect

ive

per

mea

bil

ity (

cm/s

) det

erm

ined

usi

ng P

AM

PA

Explo

rer

(pIO

N)

wit

h B

BB

lip

id m

ixtu

re m

easu

red a

t pH

= 7

.4.

dR

hodam

ine

123 u

pta

ke

was

mea

sure

d i

n M

DR

1-M

DC

KII

cel

ls u

tili

zing

Glo

max

Mult

i D

etec

tion S

yst

em (

Pro

meg

a). ‘M

DR

1 r

ecognit

ion’

was

ass

esse

d b

y m

easu

ring u

pta

ke

of

Rhodam

ine

123 i

n t

he

pre

sence

of

MD

R i

nhib

itor,

tar

iquid

ar (

5 µ

M),

30 µ

M o

f te

st a

nti

-vir

al o

r v

ehic

le, an

d c

alcu

lati

ng:

(Cav

– C

veh

)*100/(

Cta

r-C

veh

),

wher

e C

av =

conce

ntr

atio

n o

f rh

odam

ine

123 i

n t

he

pre

sen

ce o

f an

ti-v

iral

, C

veh

= c

once

ntr

atio

n i

n t

he

pre

sen

ce o

f v

ehic

le, C

tar =

conce

ntr

atio

n o

f rh

odam

ine

123 i

n t

he

pre

sen

ce o

f ta

riquid

ar. In

the

pre

sen

ce o

f ta

riquid

ar, rh

od

amin

e 123 u

pta

ke

was

1123 ±

54%

of

veh

icle

contr

ols

(n=

44)

. e H

alf-

life

in B

AL

B/c

mouse

liv

er m

icro

som

e in

cubat

ion. V

alues

are

hal

f-li

ves

cal

cula

ted f

rom

the

equat

ion T

1/2

=ln

(2)/

slope,

wher

e th

e sl

ope

was

ln(%

det

ecte

d)

agai

nst

tim

e, p

lott

ed f

rom

the

mea

n o

f at

lea

st n

=3

indep

enden

t ex

per

imen

ts. f K

inet

ic s

olu

bil

ity m

easu

red u

sing t

he

sam

e as

say m

edia

as

WE

EV

rep

lico

n a

ssay

, ex

cept

wit

h 1

0%

feta

l bovin

e se

rum

. S

ee E

xper

imen

tal

Sec

tion f

or

det

aile

d m

ethods

and s

ynth

etic

pro

cedu

res.

42

Interestingly, the N-methylated analogue CCG-212393 was ten times less active

than its parent anthranilamide CCG-211824, suggesting that the aniline nitrogen may be

engaged in internal hydrogen bonding with the ortho carbonyl oxygen, resulting in a

specific conformation favorable for activity. Further evidence for this conformational

hypothesis is provided by comparing benzylbenzamide analogue CCG-222821, with

inactive benzoylbenzamide CCG-212391. The latter would not be expected to be able to

achieve a conformation similar to the internally hydrogen bonded conformation of CCG-

211824.

Figure 12. Conformational Restriction Plan for Arene Analogs.

Analogs were designed to tether the arene rings (red, carbazole derivatives) or tie the

amide to the aniline nitrogen (blue, quinolone derivatives).

Conformational Restriction. Several rigid analogues were designed to evaluate

our conformational hypothesis (Figure 12 and Table 9). Carbazole derivatives (CCG-

222824 and CCG-222825) were intended to tether the two arenes in the molecule and

prevent rotation around the two C-N bonds. Surprisingly, these compounds were

remarkably less active than their more flexible parent, which may indicate that the

putative internal NH hydrogen bond is not sufficient for achieving the biologically active

43

conformation and that in fact the two tethered aromatic rings need to be orthogonal rather

than planar. It is also possible that tight binding to the unknown molecular target entails

induced fit, which the rigid carbazole cannot accommodate. The quinolone derivatives

(CCG-222823 and CCG-222822), on the other hand, were designed to freeze rotation

about the aniline-amide C-C bond. Their poor activity, however, indicates either a poor

match for the biologically active conformation, or a key role for the benzamide carbonyl

of CCG-211824 that has been disrupted.

Table 9. WEEV Replicon Date for Rigid Arene Analogs.

No. G IC50 (µM)a CC50 (µM)

b

222824

59.2 ± 6.5 >100

222825

18.7 ± 5.4 66.1

222823

88.3 ± 11.7 >100

222822

10.9 ± 2.0 34.1


bCell viability determined by the MTT reduction assay.

Values are mean of at least n=3 independent experiments.

44

Potential for CNS Penetration. To measure the potential for MDR1 recognition

of the arene analogs, we used the Rho123 assay.83

In Table 8, replacement of the pyrrole

of CCG-206381 with phenyl (CCG-211824) markedly increased recognition by MDR1.

Surprisingly, simple N-methylation of CCG-211824 (CCG-212393) almost completely

abated MDR1 recognition. Reductions in MDR1 recognition were also observed upon

replacement of the NH of CCG-211824 with O, C=O, or CH2 (CCG-212393, CCG-

212391 and CCG-222821, respectively) suggesting that the NH may be a key factor for

recognition. This is supported by the strong MDR1 recognition of NH homologs CCG-

222660 and CCG-222983. However, it is notable that simple removal of the aromatic

chloro group strongly attenuated MDR1 recognition (CCG-222981 and CCG-222982),

indicating that overall lipophilicity may also be a factor.

Permeability is an excellent predictor of potential BBB penetration, so selected

compounds were evaluated using pION’s BBB-PAMPA assay and PAMPA Explorer

program as previously described.83

Compounds with log Peff > 4.7 are considered to be

highly permeable, whereas those with log Peff 6 are considered poorly permeable. In

general, our compounds exhibit moderate to high BBB permeability, and all but one

(CCG-212391) possess modestly better permeability than pyrrole lead CCG-206381

(Table 8).

In vitro Metabolic Stability. Based on their WEEV replicon activities, selected

compounds were advanced into the mouse liver microsome (MLM) assay to assess their

stabilities towards Phase I oxidative metabolism (Table 8). (See Chapter V for more

information regarding metabolism.) The stability of aniline CCG-211824 was improved

with a half-life of 19 minutes, a six-fold improvement over the slightly more electron-rich

45

pyrrole CCG-206381 (3 minutes) and a two-fold improvement over the indole CCG-

205432 (9 minutes). The chlorobenzyl homologue CCG-222660 was slightly less stable

towards metabolism than CCG-211824, perhaps because of the introduction of a benzylic

carbon and increased lipophilicity. Phenolic ether CCG-212392 was as stable towards

microsomal metabolism as aniline CCG-211824. Interestingly, replacement of the NH of

CCG-211824 with CH2 (CCG-222821) markedly attenuated metabolic stability, due

either to increased lipophilicity or the creation of an additional site for oxidation.

Table 10. Antiviral Data for Select Analoguesa

FMV WEEV

CCG No. Conc. (µM) Titer (x 10

6

pfu/ml)

Viability (%

uninfected control)

Titer (x 104

pfu/ml)

DMSO NA 30.7 ± 9.0 35.6 ± 4.7 37.3 ± 7.3

205432 25 ND 69.5 ± 10.0* 1.1 ± 0.1

**

211824 25 3.7 ± 1.4* 58.1 ± 9.0 3.2 ± 2.2

*

5 10.6 ± 4.1 54.6 ± 9.2 49.2 ± 19.4

222660 25 3.2 ± 1.0**

66.6 ± 9.5* 1.6 ± 0.5

**

5 9.4 ± 5.6* 73.3 ± 9.1

* 11.7 ± 3.6

*

aAssay utilized the alphaviruses Fort Morgan Virus (FMV) and western equine

encephalitis virus (WEEV). Infections were done in cultured human BE(2)-C neuronal

cells. Viability was measure using an MTT assay, and viral titers were measured using a

plaque assay. Values are mean ± SEM of n = 3-4 independent experiments. P value

<0.05* or 0.005

** compared to DMSO control. NA, not applicable. ND, not determined.

Data from the David Miller lab.

Antiviral Activity. Based on their WEEV replicon potencies, CCG-211824 and

CCG-222660 were advanced into assays to evaluate their ability to inhibit cellular

replication of infectious live virus. Our collaborators used two parallel assays to measure

activity against infectious virus: reduction in cytopathic effect (CPE) and extracellular

46

virus titers, and examined activity against both WEEV and Fort Morgan Virus (FMV), a

WEEV-serogroup alphavirus that can be used safely under reduced biosafety level

conditions compared to WEEV. Against FMV, both CCG-211824 and CCG-222660

reduced virus titers by ten-fold when used at 25 µM, and also reduced FMV-induced CPE

similar to previous lead CCG-205432 (Table 10). Furthermore, both CCG-211824 and

CCG-222660 also reduced WEEV titers by at least 10-fold when used at 25 µM.

Although we did not do full titration curves with these experiments, CCG-222660

maintained antiviral activity against both FMV and WEEV when used at 5 µM, whereas

CCG-211824 had reduced activity at this lower concentration (Table 10).

Figure 13. Spectrum Antiviral Data for Selected Analogues.

CCG-211824 and CCG-222660 have potent and broad spectrum antiviral activity. (A)

HEK293 cells were infected with FMV at an MOI of 1 and simultaneously treated with

decreasing concentrations of the indicated compound. Virus titers in cell culture

supernatants were determined at 24 hpi by plaque assay. The horizontal dashed line

indicates FMV titers in cells treated with control DMSO. Calculated IC50 values for

CCG-205432, CCG-211824, and CCG-222660 were 1.0 ± 0.5, 5.2 ± 1.0, and 2.3 ± 1.2

µM, respectively. (B) HEK293 cells were infected with the indicated viruses at an MOI

of 0.1, simultaneously treated with the indicated compound at 25 µM, and virus titers in

tissue culture supernatants were determined by plaque assay at 24 hpi. P <0.05* or

0.005** compared to DMSO-treated controls. Data from the David Miller Lab.

47

To further evaluate the antiviral potency and breadth of activity for CCG-211824

and CCG-222660, we completed both full titration studies with FMV in HEK293 cells

(Figure 13A) and examined their inhibitory activity against viruses derived from three

different families: Venezuelan equine encephalitis virus (VEEV, TC-83 vaccine strain;

Togaviridae), California encephalitis virus (CEV; Bunyaviridae), and

encephalomyocarditis virus (EMCV; Picornaviridae) (Figure 13B). Both CCG-211824

and CCG-222660 had dose-dependent antiviral activity against FMV, where CCG-

222660 had an IC50 similar to previous lead CCG-205432 (Figure 13A). In addition,

both CCG-211824 and CCG-222660 had demonstrable antiviral activity against VEEV,

CEV, and EMCV in cultured cells, with an approximate 10-fold reduction in infectious

virus titers for all three pathogens, similar to previous lead CCG-205432 (Figure 13B).

Mechanism of Action Studies. The use of the WEEV replicon assay, a

phenotypic cell-based assay, increases the probability of selecting compounds having a

cellular rather than a viral target for antiviral activity. There are several advantages of

targeting host factors over viral proteins. The most significant advantage is a decreased

probability of the targeted virus developing resistance to therapeutics.105

Our

collaborator’s studies with the lead CCG-205432 and related carboxamide analogs

indicated that the mechanism of action for their antiviral activity is through the

modulation of cellular cap-dependent translation.104

To examine whether our monocyclic

analogs used a similar mechanism, we evaluated the ability of CCG-211824 and CCG-

222660 to modulate cellular cap-dependent translation (Figure 14A). For these

experiments, we used BSR-T7 cells and two reporter plasmids: pSV40-LUC, which

produces a capped mRNA through nuclear transcription and export, and pCITE-LUC,

48

which produces an uncapped mRNA containing a cap-independent translational element

(CITE) through cytoplasmic transcription that is driven by a T7 promoter (Figure

14B).106

Similar to lead CCG-205432 and previous generation compounds, both CCG-

211824 and CCG-222660 had no effect upon cap-independent translation, but potently

inhibited cellular cap-dependent translation of transcripts produced by the pSV40-LUC

reporter plasmid (Figure 14B). These results suggest that the anthranilamide analogs

CCG-211824 and CCG-222660 also function as antiviral compounds, in part, through

suppression of host cell cap-dependent translation.

Figure 14. Effect of Selected Analogs on Transfected Gene Expression in BSR-T7

Cells.a

9a and 11a modulate cellular gene expression. BSR-T7 cells were transfected with the

expression plasmids pSV40-LUC (circles) or pT7/CITE-LUC (triangles), treated with

decreasing concentrations of the indicated compound, and fLUC activity was measured

20 h later. Calculated IC50 values for suppression of pSV40-LUC activity for 9a and 11a

were 2.2 ± 0.2 and 1.0 ± 0.1 µM, respectively. 1 has an IC50 value of 0.1 µM in this

assay.104

N=3 or 4 for all data, and results are the mean ± SEM. Data from the David

Miller lab.

49

Synthesis

The piperidine-containing pyrrole analogs CCG-204054, CCG-206381, and

CCG-208914 and the imidazole analogs CCG-206586, CCG-208913, and CCG-208916

were first prepared by alkylating the appropriate azole with 4-chlorobenzyl chloride to

provide the methyl ester. Saponification of this ester and amide coupling with ethyl

isonipecotate gave the intermediate. This ethyl ester was then hydrolyzed and coupled

with the appropriate amine (Scheme 7).

Scheme 7. Preparation of Pyrrole and Imidazole Analogs.

Reagents and conditions: (a) p-chlorobenzyl chloride, K2CO3, DMF, 60 °C, 36 h; (b) 7M

NaOH, EtOH, 70 °C, 4 h; (c) 26, EDCHCl, HOBt, DIPEA, DMF, rt, 24 h; (d) 7M

NaOH, EtOH, 60 °C, 8 h; (e) 27, EDCHCl, HOBt, DIPEA, DMF, rt, 24 h.

50

Scheme 8. Preparation of Azetidine Analogs.a

aReagents and conditions: (a) methyl 4-azetidinecarboxylate hydrochloride, EDC, HOBT,

DIPEA, DCM, rt; (b) 10% aq. NaOH, EtOH; (c) R-NH2, EDC, HOBT, TEA, DCM, rt.

The azetidine-containing pyrroles CCG-208793, CCG-208829, and CCG-

208915 were prepared through the union of N-benzyl pyrrole 28 and methyl azetidine-3-

carboxylate, followed by base hydrolysis and amidation with the appropriate amine

(Scheme 8). Urea analogs CCG-208825, CCG-208826, CCG-208827, and CCG-

208828 were synthesized by the addition of ethyl isonipecotate 26 to isocyanates 30a,b,

followed by hydrolysis and amidation with the appropriate amine (Scheme 9). The

acyclic amides CCG-208846, CCG-208847, CCG-208848, and CCG-208849 were

synthesized from the amide coupling of the appropriate carboxylic acid 32a,b and ethyl

isonipecotate 26, followed by hydrolysis/amidation with the appropriate amine similar to

the previous syntheses (Scheme 9).

51

Scheme 9. Preparation of Acyclic Amide and Urea Analogs.a

aReagents and conditions: (a) 26, DCM, rt; (b) 10% aq. NaOH, EtOH, rt; (c) R-NH2,

EDC, HOBT, TEA, DCM, rt; (d) 26, EDCHCl, HOBT, TEA, DCM, RT.

The right-hand portion of the pyridine-bearing analogs, N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide dihydrochloride 36 (Scheme 10), was assembled by

first boc-protecting ethyl isonipecotate 26. The protected ethyl ester was then hydrolyzed

to the carboxylic acid and coupled with 4-(2-aminoethyl)pyridine 27. The protecting

52

group was then removed under dry acidic conditions to yield the dihydrochloride salt 36

of the desired amine.

Scheme 10. Preparation of the Right-Hand Amine Dihydrochloride 36.a

aReagents and conditions: (a) Boc2O, Na2CO3, H2O, THF, reflux; (b) NaOH, H2O, EtOH,

RT; (c) 4-(2-aminoethyl)pyridine, EDCHCl, HOBT, DCM, RT; (d) HCl, dioxane, RT.

N-benzylpyrrolidine enantiomers CCG-211757 and CCG-211758 were prepared

through base-catalyzed alkylation of proline with 4-chlorobenzylchloride to protect

stereochemical integrity via the carboxylate anion,107

followed by amide coupling with

amine dihydrochloride 3683

to yield the appropriate (R)- and (S)- analogs (Scheme 11).

Pyridine-bearing enantiomers CCG-211754 and CCG-211756, and indane-bearing

enantiomers CCG-211753 and CCG-211755, were synthesized in similar fashion, first

by acylating proline under classical Schotten-Baumann conditions108, 109

followed by

amide coupling with ethyl isonipecotate 26 (Scheme 11). Hydrolysis of the ethyl ester

(R)- or (S)-40 through a non-racemizing protocol110

provided the carboxylic acid, and

amidation with amine 27 gave the desired (R)- and (S)- benzoyl analogs.

53

Scheme 11. Preparation of N-Benzyl and N-Benzoyl Pyrrolidine Analogs.a

aReagents and conditions: (a) 2a, KOH, IPA, 40 °C; (b) 36, HATU, TEA, DCM, RT; (c)

4-chlorobenzoylchloride, IPA, KOH, H2O, 0 °C; (d) 26, HATU, TEA, DCM, RT; (e) 9:1

DCM:MeOH, NaOH, rt; (f) 27, HATU, TEA, DCM, RT.

Salicylamide analogue CCG-212392 was prepared from the Chan-Lam

coupling111

of methyl salicylate 41 and 4-chlorophenyl boronic acid, providing the ether

intermediate 42 in low yield, followed by ester hydrolysis and amidation (Scheme 12).

Secondary phenyl anthranilamides CCG-211824 and CCG-222981 were assembled from

Buchwald amination112, 113

of triflated methyl salicylate and the appropriate aniline,

followed by saponification/amidation. The N-methyl anthranilamide derivative CCG-

54

212393 was accessed through methylation of coupling intermediate 43a, followed by the

usual saponification and amide coupling.

Scheme 12. Preparation of Anthranilamide and Salicylamide Analogues.a

aReagents and conditions: (a) Cu(OAc)2, 4-chlorophenylboronic acid, TEA, pyridine,

DCM, rt; (b) 10% aq. NaOH, EtOH, RT; (c) 36, EDC, HOBT, TEA, DCM, RT; (d)

PhN(Tf)2, TEA, DMF, RT; (e) 4-chloroaniline or aniline, Pd(OAc)2, DPPP, Cs2CO3,

toluene, reflux; (f) ethyl isonipecotate, EDC, HOBT, TEA, DCM, RT; (g) 4-(2-

aminoethyl)pyridine, EDC, HOBT, TEA, DCM, rt; (h) MeI, Cs2CO3, DMF, RT.

55

Benzyl anthranilamides CCG-222660 and CCG-222982 were obtained from the

alkylation of methyl 2-aminobenzoate 46 and the corresponding benzyl halide, followed

by the base hydrolysis and amidation (Scheme 13).

Phenethyl anthranilamide CCG-222983 could not be prepared through direct N-

alkylation of methyl 2-aminobenzoate because the electrophile was highly prone to

elimination. Conveniently, the transformation could be achieved through a Jourdan-

Ulmann coupling114

of 2-bromobenzoic acid 48 and 1-(2-aminoethyl)-4-chlorophenyl

amine in ethylene glycol dimethyl ether (Scheme 14). Base hydrolysis and amidation

then provided the desired analog.

Scheme 13. Preparation of Benzyl Anthranilamide Analogues.a

aReagents and conditions: (a) 4-chlorobenzyl chloride or benzyl bromide, tBuOK or

Cs2CO3, DMF; (b) 10% aq. NaOH, EtOH, rt; (c) 36, EDC, HOBT, TEA, DCM, rt.

Scheme 14. Preparation of Phenethyl Anthranilamide Analogue CCG-222983.a

aReagents and conditions: (a) 1-(2-aminoethyl)-4-chlorophenyl amine, Cu2O, Cu

0,

K2CO3, EDME, 130 °C; (b) 36, EDC, HOBT, TEA, DCM, rt.

56

Scheme 15. Preparation of Benzyl and Benzoyl Benzoate Analogues.a

aReagents and conditions: (a) AlCl3, PhCl, reflux; (b) 36, EDC, HOBT, TEA, DCM, rt;

(c) Zn0, CuSO45H2O, 28% aq. NH3, reflux.

The preparation of benzyl- and benzoyl benzamide derivatives (CCG-222821 and

CCG-212391 respectively) entailed the known Friedel-Crafts acylation of chlorobenzene

with phthalic anhydride, 115

which delivered ketone intermediate 51 from which a simple

amidation with 36 provided the benzoylbenzamide analogue CCG-212391 (Scheme 15).

The related benzylbenzamide analogue CCG-222821 was accessed through a dissolving

metal reduction of the ketone intermediate 51116, 117

to afford benzyl benzoic acid 52,

which was followed by amidation with amine 36. Interestingly, the ketone could not be

reduced without concomitant reduction of the chloride under several heterogenous

hydrogenation conditions.115, 118

Also, attempts to reduce the ketone to the respective

alcohol with NaBH4 failed,119

even with excess borohydride and vigorous conditions.

Thionation with Lawesson’s Reagent and subsequent Raney-Ni reduction also could be

57

used to generate the desired benzyl intermediate 52, but the dissolving metal reduction in

Scheme 15 was found to be more direct and efficient.

Scheme 16. Preparation of Carbazole Analogues.a

aReagents and conditions: (a) pyrrolidine, 37% aq. HCHO, EtOH, reflux; (b) s-BuLi,

THF, CO2(g), then 1 M aq. HCl, reflux; (c) sat. methanolic HCl, reflux; (d) SO2Cl2, DCM,

0 °C; (e) 10% aq. NaOH, EtOH, THF, rt; (f) 36, EDC, HOBT, TEA, DCM, rt.

Carbazole analogues were synthesized as shown in Scheme 16. Ortho-directed

lithiation of carbazole 53 followed by carboxylation with carbon dioxide in a manner

similar to that reported by Katritzky provided key intermediate 54.120

Direct coupling

with amine 36 provided analogue CCG-222824. The carbazole carboxylic acid 54 could

not be chlorinated with NCS under a variety of conditions,121, 122

while the methyl ester

yielded a complex mixture of variously chlorinated carbazoles. Ultimately, sulfuryl

chloride123

gave clean regioselective conversion to chloro-substituted carbazole ester 55

as confirmed by 1HNMR. This ester was then hydrolyzed to the corresponding carboxylic

acid and coupled with amine 36 to give the desired analogue CCG-222825.

58

Scheme 17. Preparation of Quinolone Analogues.a

aReagents and conditions: (a) 6 M aq. HCl, 1,4-dioxane, reflux; (b) ethyl isonipecotate,

NaI, Na2CO3, DMF; 80 °C; (c) 4-chlorophenylboronic acid, Cu(OAc)2, TEA, pyridine,

DCM, rt; (d) 10% aq. NaOH, EtOH, THF, rt; (e) 4-(2-aminoethyl)pyridine, EDC, HOBT,

TEA, DCM, rt; (f) 4-chlorobenzyl chloride, KOtBu, DMF, 0 °C to rt.

Finally, quinolone analogues were accessed as shown in Scheme 17. Hydrolysis

of 2,4-dichloroquinoline 56124, 125

and subsequent addition/elimination with ethyl

isonipecotate 26 gave intermediate 57 which subsequently could be N-arylated via a

Chan-Lam coupling,126

then saponified and coupled with amine 27 to give

phenylquinolone CCG-222823. Conversely, 57 could be alkylated with 4-chlorobenzyl

59

chloride 2a, hydrolyzed and coupled under similar conditions to the phenylquinolone to

provide the benzylquinolone CCG-222822.

Conclusion

The primary goal of this chapter was the reduction of molecular weight, as low MW

is associated with improved BBB-permeability and is a feature of successful CNS drugs.

To this end, we successfully developed a novel MDR1-evasive pyrrole compound (CCG-

206381) with sub-micromolar potency and demonstrable in vivo CNS penetration. This

marks the first successful alphavirus antiviral with measurable CNS exposure, and

confirmed our predictions that reduction of MW was a viable tactic in achieving

favorable CNS permeability. However, this compound was remarkably susceptible to

CYP-mediated metabolism; therefore, an additional goal was improvement of metabolic

stability, and we identified the aniline moiety as a suitable substitute for pyrrole. We

unveiled phenylaniline CCG-211824 and benzylaniline CCG-222660 as low-to-sub-

micromolar inhibitors with MLM half-lives twice that of the original indole lead CCG-

205432 and five-to-six times that of the pyrrole lead CCG-206381. Importantly, these

aniline compounds demonstrated excellent activity (almost two-fold titer reduction)

against live viruses from three different families. This is an important step towards the

development of antivirals that are both potent and bioavailable.

60

Conformational Restriction Chapter IV.

Rationale

Potency is important for many aspects of drug delivery; potent compounds are

generally more amenable to oral administration because they require less material to

achieve the desired therapeutic effect, and they are less prone to off-target effects

(toxicity) because of the low doses required.79

Therefore, as with any drug, a high

therapeutic index, representing high potency and low toxicity, is a major goal for WEEV

inhibitors. Potency is also especially important for the successful action of many CNS-

active drugs; due to the brain-blood barrier (BBB), very little of the available drug will

actually penetrate the brain, as described later.

An important task in potency optimization is the identification of the bioactive

conformation of the inhibitors.127-130

In the absence of structural information, this can

only be accomplished by synthesizing and evaluating rigid analogs.131, 132

Of the multiple

conformations that a drug can adopt, only a small subset will have maximal activity if the

binding site is well defined and not highly flexible.128, 133

It is therefore informative to

lock out or freeze certain conformations and observe their effect on activity in an effort to

pinpoint the active one, especially when the binding site is unknown.134

It is also a very

useful strategy for improving selectivity versus off-target effects.135-137

When designing

such analogs, it is important to minimize the number of additional atoms used in locking

a conformation because adding molecular weight or introducing new interactions with the

61

binding site may have their own effects on activity, thereby confounding interpretation of

the actual impact of conformational restriction. Rigid analogs may be prepared utilizing

several approaches to restricting rotatable bonds: a) substituting small functionality with

sterically demanding functionality; b) bridging nonadjacent atoms; and c) restricting ring

conformations.

Figure 15. Steric Approach to Conformational Restriction.

Installation of sterically restricting functionality is most often the simplest

approach of the three tactics and retains some degree of flexibility. In the case of lead

CCG-102516, the N-benzyl encounters minimal resistance to rotation from the small

indole hydrogens and would be expected to adopt a configuration that minimizes steric

clashes with the amide carbonyl (Figure 15 A). On the other hand, methylation of the

indole 7-position hinders its ability to rotate over this region and would be expected to

62

greatly limit the available conformers, inducing a conformation (63) that is distinct from

61 (Figure 15 B).

Figure 16. Tethering Approach to Conformational Restriction.

The second tactic, bridging of nonadjacent atoms, is a much more restrictive

approach. For example, the distal amide of CCG-102516 is free to rotate about

piperidine-amide C-C bind (Figure 16), but inclusion of a bridging methylene between

the piperidine 3-position and the amide nitrogen provides a compound with an

immobilized carboxamide conformer.

Figure 17. Ring-Locking Approach to Conformational Restriction.

63

The third approach, the locking or steric restriction of ring conformers, is similar

to that of the previous tactic in that it most frequently involves bridging of nonadjacent

and (often) transannular atoms (Figure 17). The lead compound CCG-102516 features a

1,4-disubstituted piperidine, and due to the meso C2 symmetry about the ring, there

predominantly exist two major conformers: equatorial-equatorial (66) and axial-

equatorial (67). Either of these can be conveniently locked. An ethylene bridge across the

piperidine 2- and 6-positions can only axial-axial (68), resulting in a locked equatorial-

equatorial relationship between the piperidine substituents. Similarly, preparation of the

fused cyclopropane/pyrrolidine (69) allows access to the axial-equatorial analog, with

minimal impact on molecular weight.

SAR

Initial SAR focused on the restricting the indole and benzamide portions of the

lead CCG-102516. Biological results for indole core analogs appear in Table 11.

Utilizing the steric approach to conformational restriction, two compounds were designed

on the notion that strategically placed methyl groups would impede bond rotations of

interest while only contributing a small increase in molecular weight. Both the 7-

methylindole CCG-206447 and the 3-methylindole CCG-205431 possessed substantially

reduced potency, indicating that the methyl groups may in fact be blocking access to the

active conformations.

64

Table 11. WEEV Replicon Data for Methylindole Analogs.

CCG No. L M-R IC50 (µM)a CC50 (µM)

b

102516†

15.6 ± 1.8 >100

205431

>100 >100

206447

61 ± 39 >100

206485

17.1 ± 1.3 >100




†Synthesized by Janice Sindac.

65

Table 12. WEEV Replicon Data for Rigid Benzamide Analogs.


b

102516†

15.6 ±1.8 >100

205470

>100 >100

205471

>100 >100

205472

>100 >100

205473

>100 >100

205474

>100 >100

206328

>100 >100

206382

0.53 ± 0.04 >100

206549

1.7 ± 0.1 >100

206550

7.1 ± 0.9 >100

206397†

15.2 ± 4.7 62 ± 13

206587

>100 >50

206382

0.53 ±0.04 >100





66

Restricted of the Terminal Carboxamide. We then synthesized and evaluated

analogs of the benzamide featuring phenylpyrrolidines, isoindolines, indolines, and

indane substructures (Table 12) and a bicyclic compound CCG-206485 (Table 11), but

the majority of analogs exhibited no activity, which was not entirely undesirable.

Uniformly active compounds would indicate a non-selective binding site – one to which

binding optimization would be difficult. Excitingly, one compound (CCG-206382)

possessed significantly better activity than others in this series, and constituted the most

active compound in the analog arsenal at that time. This result was most likely not the

product of increased lipophilicity or chain extension because analogs of similar logP

(CCG-206550) and length (CCG-206587) have higher IC50 values (Table 12).

Interestingly, the racemic methyl analog of CCG-206382 (CCG-206549) was also

active, though just over two-fold less so than its more potent relative. Curiously, CCG-

206550 was similarly active to the lead CCG-102516, but CCG-205472 was completely

inactive despite being shorter by only a single methylene unit. Overall, the results in

Table 12 suggest that the amide binding pocket of the unknown molecular target is very

well defined and discriminating.

Indane SAR. Due to the superb in vitro potency of CCG-206382 in the WEEV

replicon assay, the indane moiety was incorporated into other WEEV inhibitor analogs,

including those of the low molecular weight class (Chapter III). As previously discussed

in that chapter, Janice Sindac had discovered the equally potent compound CCG-205432

bearing a pyridine in place of the indane. Curiously, related analogs differing only by the

indane or pyridine functionality did not possess similar activities (Table 13); in some

cases, the IC50 values differed in excess of one hundred-fold (e.g. CCG-206381

67

compared to CCG-208914, and CCG-209021 compared to CCG-209020.) The indane

compounds also behaved differently than their pyridine counterparts in the replicon

assay. For example, an IC50 value for CCG-209793 could not be deduced because the

concentration-inhibition plot was uncharacteristically linear, and more than one

compound’s plot oddly plateaued in a manner inconsistent with every other analog and

class synthesized to-date. These results questioned the validity of CCG-206382’s

activity, but our collaborators found no evidence of direct luciferase inhibition. The

compound was consequently promoted for further studies to evaluate its true efficacy

against live virus.

Table 13. WEEV Replicon Data Comparisons of Analogs Based on CCG-205432

and CCG-206382.

CCG # R IC50 (µM)a CC50 (µM)

b

205432†

0.53 ± 0.08 65.0

206382

0.53 ± 0.04 >100

206381

0.68 ± 0.04 >100

208914

61.8c 70.3

208916

>100 >100

208913

>100 >100

208915

22.0 ± 8 >100

208793

CNBDd >100

68

Table 13. Continued.

CCG # R IC50 (µM)a CC50 (µM)

b

209021

0.93 ± 0.2 8.0

209020

>100 >100

211756

>100 >100

211753

>100 >100

211754

>100 >100

211755

>100 >100

208825

60 ± 5 >100

208826

>100 >100

208827

41 ± 5 >100

208828

>100 >100

208846

>100 >100

208847

>100 >100

208848

>100 >100

208849

33 ± 15 >100



by the MTT reduction assay. Values are mean of at least n=3 independent experiments. cValue is n=1 experiment.

dCould not be determined.


69

Antiviral Activity. When cells infected with WEEV or NSV (neuro-adapted

sinbis virus) were treated with the pyridine analog CCG-205432, viral titer was reduced

by nearly two log units, and cell viability was significantly improved (Figure 18).

However, the indane analog CCG-206382 (despite being equipotent in the WEEV

replicon assay) neither decreased viral titer nor improved cell viability relative to the

negative control, suggesting that the compound was truly not active. Analogs featuring

the indane moiety were subsequently dropped.

Figure 18. Activity of Various Analogs Against Live Virus.

Data from the David Miller Lab.

70

Piperidine Amide SAR. Having determined the terminal carboxamide portion of

the analogs to be a challenging site for conformational restriction, our attention was

turned towards the central piperidine ring. As in Chapter III (low molecular weight

series), CCG-205432 was employed as the lead due to its potency and demonstrable

activity against live virus, and thus this series features the right-hand pyridylethyl amide.

Biological results appear in Table 14. In Chapter II (reduced TPSA series), it was noted

that there appeared to be a limited tolerance for TPSA reduction, perhaps because certain

structure-activity requirements were not met. It was noted that compounds like CCG-

211826 (Table 2), despite being structurally similar to potent leads, lacked the right-hand

secondary amide. To evaluate the postulated importance of this functionality, four

compounds were prepared (Table 14). Activities of the inverse amide CCG-2120532 and

the shifted amide CCG-224001 are over 30-fold less than CCG-205432, confirming that

the amide is important to the unknown pharmacophore. In particular, both of these

analogs retain the amide carbonyl in the same relative location, but differ in the location

of the amide nitrogen, which indicates that the position of the carbonyl is apparently

more important than the NH. The amide carbonyl may be engaged as a hydrogen-bond

acceptor, and, due to the dependence on the acceptor-donor angle, translocation of the

carbonyl by one methylene unit may be sufficient to deprive the molecule of a productive

hydrogen bond in the unknown target, accounting for the diminished activity.

Furthermore, complete excision of the amide (e.g. E- and Z-alkenes CCG-211823 and

CCG-211825) returned analogs with no activity, complementing this hypothesis.

71

Table 14. WEEV Replicon Data for Central Piperidine Analogs.


b

205432†

0.53 ± 0.08 65.0

212052

17.6c 66.7

c

224001

24.7 ± 3.5 77.3

211823

>50 >50

211825

>50 >50

212390

24.4 ± 4.0 78.0

211822

3.9 ± 0.4 74.2

211829

>50 >50

211761†

1.3 ± 0.4 63.2

72

Table 14. Continued.


b

212394

24.4 ± 6.3 61.0

222980

24.6 ± 1.6 >100

224000

0.96 ± 0.06 50.9

224220

10.9 ± 0.6 56.5

224002

73.1 ± 29.1 88.1

222661

0.49 ± 0.09 41.1



by the MTT reduction assay. Values are mean of at least n=3 independent experiments. cValue is n=1 experiment.


Core Piperidine SAR. The significantly reduced activity of the urea analog

CCG-212390 and eneamide CCG-211822 are also consistent with the postulated

importance of the secondary amide (Table 14). Both analogs were built to rigidify and

flatten the core ring, and such modifications may have changed the projection of the

amide carbonyl. The bicyclic analog CCG-212394 may display the amide in a similarly

nonproductive manner, especially in light of the better activity of the very similar (but

73

less restricted) analog CCG-211761. The α-methyl analog CCG-211829 was much less

active, presumably because the methyl was either impeding favorable chair

conformations or occluding the amide carbonyl. The exo-bicycle CCG-222980 was also

less potent than the lead. Interestingly, the ethylene-bridged piperidine CCG-224000 was

a mere two-fold less potent than CCG-205432, suggesting that the locked equatorial-

equatorial relationship between the 1- and 4-substituents may be indicative of the

unknown bioactive conformation.

The importance of length to the activity of WEEV inhibitors was discussed in

Chapter II and Chapter III, and, unsurprisingly, the azetidine-containing CCG-224002

was less active than its piperidine-containing counterpart CCG-205432 (Table 14). To

determine the effect of larger rings on activity, the azapane analog CGG-222661 was

synthesized and tested. Interestingly, this compound was equally potent with the lead

despite being less rigid and possessing an offset right-hand amide. Furthermore, the

active enantiomer could potentially be two-fold more active than the racemate, marking

this compound as the most active to-date in the WEEV replicon assay.

Synthesis

Restricted analogs based upon the benzylamide lead CCG-102516 were generally

prepared through amide coupling of various amines with the core carboxylic acid 71,

which was itself accessed via coupling of the indole-2-carboxylic acid 4a with ethyl

isonipecotate 26 followed by saponification (Scheme 18).

74

Scheme 18. General Preparation of Rigid Benzamide Analogs.a

aReagents and conditions: (a) 26, EDC∙HCl, HOBT, TEA, DCM, RT; (b) 10% aq.

NaOH, EtOH, RT; (c) HNR1R2, EDC∙HCl, HOBT, TEA, DCM, RT.

Scheme 19. Preparation of 2-(aminomethyl)indane 75.a

aReagents and conditions: (a) HMDS, HATU, DIPEA, DMF, rt, 20 h; (b) LiAlH4, THF,

RT, 8 h.

2-(Aminomethyl)indane 75 (Scheme 19, for the synthesis of CCG-206382) was

accessed first through amidation with the 2-indane carboxylic acid 73 and HMDS, after

which aqueous workup conditions resulted in hydrolytic degradation of the silyl

functionality to reveal the primary carboxamide 74. This procedure was found to be more

efficient than direct aminolysis of the carboxylic acid. Reduction of the carboxamide with

LAH provided the desired amine 75, which was coupled with indole carboxylic acid 71

as described in Scheme 18. Racemic 2-(2-aminoethyl)indane 78 was prepared first

75

through double displacement of dibromoxylene 76 with acetylacetate followed by

reductive amination of the ketone 77 (Scheme 20).

Scheme 20. Preparation of Racemic 2-(2-Aminoethyl)indane.a

aReagents and conditions: (a) ACAC, NaH, DMF; (b) 7 M NH3/MeOH, NaBH4, RT, 20

h.

The 7- and 3-methyl indole analogs were generally prepared through classical

Fischer indolization (Scheme 21 and Scheme 22). The appropriate anilines (o-toluidine

79 and aniline 84) were transformed into their respective diazonium salts and reduced to

hydrazines through the action of tin(II) chloride.138

The hydrazines were then allowed to

undergo Fischer indolization with the necessary β-ketoester 80 or 85, but only the 7-

methyl indole required a transition metal Lewis acid catalyst to facilitate cyclization to

indole 81. Overall, this route was found to be more expedient than the reported two-step

Japp-Klingemann/Fischer indolization approach139

to the indole scaffold. Alkylation of

the indole N1-position with 4-chlorobenzyl chloride 2a was found to be slow for the 7-

methyl analog 81, presumably due to steric hindrance of the nucleophile. Subsequent

saponification and amide coupling with benzylamine yielded the desired methyl indole

analogs CCG-205431 and CCG-206447.

76

Scheme 21. Preparation of 7-Methylindole CCG-206447.a

aReagents and conditions: (a) 79, NaNO2, conc. HCl, 0 °C, 30 min; then SnCl2, 4 °C, 11

h, 56%; (b) 80, ZnCl2, TsOH, EtOH, reflux, 8 h; (c) 2a, K2CO3, DMF, 60 °C, 3 d, 46%;

(d) 10% aq. KOH, EtOH, 70 °C, 4 h, 94%; (e) N-benzylpiperidine-4-carboxamide,

EDCHCl, HOBT, DIPEA, DMF, RT, 18 h, 47%.

Scheme 22. Preparation of 3-Methylindole CCG-205431.a

aReagents and conditions: (a) PhNH2, NaNO2, conc. HCl, 0 °C, 15 min; then SnCl2, 0 °C,

24 h; then 85, EtOH, TsOH, reflux, 12 h, 65%; (b) 2a, K2CO3, DMF, 60 °C, 12 h, 72%;

(c) THF, 5 M aq. NaOH, 55 °C, 18 h, 96%; (d) N-benzylpiperidine-4-carboxamide

hydrochloride, HATU, DIPEA, DMF, RT, 20 h, 51%.

77

The pyrrolopiperidine analogs CCG-206485 and CCG-212394 were accessed

first through the partial reduction of the imide 88 to the amide 89 as reported by Dugar et

al.140

Presumably, selectivity arises from chelation-directed reduction of the carbonyl

situated on the same side of the molecule as the azine nitrogen.141

Complete reduction of

the pyridine gave the piperidine 90, and amidation with this and the indole carboxylic

acid 4a provided intermediate 91, which was alkylated with the requisite electrophiles

(Scheme 23).

Scheme 23. Preparation of pyrrolidopiperidine Analogs CCG-206485 and CCG-

212394.a

aReagents and conditions: (a) Zn

0, AcOH, 6 h, 115 °C, 38%; (b) PtO2, 50 psi H2, AcOH,

RT, 2 d; (c) 4a, EDCHCl, HOBT, DIPEA, DCM, RT, 12 h, 56%; (d) Ar(CH2)nBr, NaH,

DMF, RT, 18 h, 72%.

78

Scheme 24. Preparation of Inverse Amide CCG-212052.a

aReagents and conditions: (a) EDCHCl, HOBT, TEA, DCM, RT, 30 h, 71%; (b) HCl,

dioxane, RT, 1 h, 100%; (c) 4a, EDCHCl, HOBT, TEA, DCM, RT, 22 h, 61%.

Scheme 25. Preparation of Shifted Amide CCG-224001.a

aReagents and conditions: (a) EDCHCl, HOBT, TEA, DCM, RT; (b) KOH, H2O, EtOH,

RT; (c) 4-(aminomethyl)pyridine, EDCHCl, HOBT, TEA, DCM, RT.

The inverse amide CCG-212052 was prepared through the amide coupling of N1-

protected 4-aminopiperidine 92 and 3-(4-pyridyl)propanoic acid 93. Subsequent

79

deprotection and coupling with indole carboxylic acid 4a furnished the desired inverse

amide CCG-212052 (Scheme 24).

To prepare the shifted amide analog CCG-224001, the commercially available

amino ester 95 was first coupled with the indole carboxylic acid 4a. The ester of the

resulting intermediate was then hydrolyzed and coupled with 4-(aminomethyl)pyridine to

provide the final compound (Scheme 25).

It was anticipated that a Wittig reaction would ultimately provide the key (Z)-

alkene feature of analog CCG-211829, so the requisite phosphonium bromide 98 was

prepared from 4-(3-hydroxypropyl)pyridine 97.142

This was then deprotonated by nBuLi

to provide the ylide, which was subsequently permitted to react with the piperdinyl

aldehyde 8 to provide the desired alkene 99a with >50 Z:E selectivity. Deprotection and

subsequent coupling with the indole carboxylic acid 4a gave the (Z)-alkene analog CCG-

211829 (Scheme 26).

A large number of highly E-selective olefinations exist,143

but because the

phosphonium bromide salt was already in hand, the Schlosser modification of the Wittig

reaction was employed in order to access (E)-alkene analog CCG-211822 (Scheme 26).

In this procedure, the intermediate oxaphosphetane is deprotonated by PhLi – uniquely

suited to deprotontion of sterically shielded protons144

– and allowed to equilibrate to the

more thermodynamically favorable trans-betaine carbanion before being quenched with

tBuOH. In this way, the desired E stereochemistry was quickly accessed to provide the

(E)-alkene 99b. Deprotection and amide coupling with indole carboxylic acid 4a yielded

the desired (E)-alkene analog CCG-211822.

80

Scheme 26. Preparation of (E)- and (Z)-alkene Analogs.a

aReagents and conditions: (a) Conc. HBr, PPh3, tol, reflux, 11 h, 34%; (b) n-BuLi, THF,

RT; then 8, RT, 12 h, 52%; (c) 4 M HCl/1,4-dioxane, RT, 1 h; (d) 1-(4-chlorobenzyl)-

1H-indole-2-carboxylic acid, EDCHCl, HOBT, TEA, DCM, RT, 14 h, 56% (2 steps); (e)

PhLi, LiBr, THF, RT, 20 min; then 8, THF, -78 °C, 30 min; then PhLi, -78 °C, 30 min;

then t-BuOH, THF, -78 °CRT, 36 h, 41%; (f) 4 M HCl/1,4-dioxane, RT, 1 h; (g) 1-(4-

chlorobenzyl)-1H-indole-2-carboxylic acid, EDCHCl, HOBT, TEA, DCM, RT, 14 h,

60% (2 steps).

The piperazinyl urea analog CCG-212390 was prepared by treating boc-

piperazine 100 with phosgene and 4-(2-aminoethyl)pyridine 27 (Scheme 27). Order of

addition was critical; if 27 was treated with phosgene, the intermediate N-acyl chloride

would rapidly react with the pyridine nitrogen and no productive chemistry would arise.

Thus, the boc-piperazine 100 was treated first with phosgene, and the pyridyl amine 27

was added last. Once the urea was in hand, deprotection and coupling with indole

carboxylic acid 4a provided the desired urea analog CCG-212390.

81

Scheme 27. Preparation of Urea Analog CCG-212390.a

aReagents and conditions: (a) COCl2, TEA, tol, DCM, 0 °C, 1 h, 81%; (b) HCl, dioxane,

RT, 1 h, 100%; (c) 4a, EDCHCl, HOBT, TEA, DCM, RT,

Initial attempts to access the tetrahydropyridine core of CCG-211823 proceeded

through the partial reduction of the formyl pyridinium salt of ethyl isonicotinate with

NaBH4,145, 146

but this method was found to be poor yielding and complicated by side

products. The selenoxide elimination was then identified as a viable alternative, shown in

Scheme 28.147, 148

Treatment of protected ethyl isonipecotate 102 with LDA and

subsequent addition of phenylselenyl chloride provided the phenylselenide 103. The

compound could undergo the oxidation-elimination at this point, but it was found that

carrying the selenyl functionality through the first amidation aided purification efforts. In

this way, phenylselenide intermediate 105 was oxidized with mCPBA under cold

conditions and basified to prevent Bronsted-acid catalyzed hydrolytic decomposition via

a seleno-Pummerer pathway,149

and the resulting tetrahydropyridine ethyl ester 106 was

82

saponified then amidated with 4-(2-aminoethyl)pyridine to give the desired analog CCG-

211823.

Scheme 28. Preparation of Tetrahydropyridine Analog CCG-211823.a

aReagents and conditions: (a) Boc2O, Na2CO3, THF, H2O, reflux, 86%; (b) n-BuLi,

DIPA, -78 °C, 15 min; then PhSeCl, THF, -78 °CRT, 3 h, 58%; (c) 4 M HCl/1,4-

dioxane, RT, 2 d, 100%; (d) 4a, EDCHCl, HOBT, TEA, DCM, RT, overnight, 63%; (e)

mCPBA, DCM, -78 °C, 30 min; then TEA, DCM, -78 °C; then -78 °CRT, 1 h, 82%; (f)

10% aq. NaOH, EtOH, RT, 95%; (g) 27, EDCHCl, HOBT, TEA, DCM, RT, 91%.

In a manner similar to the preparation of the tetrahydropyridine analog CCG-

211823, Boc-protected ethyl isonipecotate 102 was alkylated with methyl iodide after

treatment with LDA to provide the methylpiperidine 107. This piperidine was

deprotected and coupled with indole carboxylic acid 4a, and the ethyl ester 109 was

83

saponified and coupled with 27 to give the desired methyl analog CCG-211829 (Scheme

29).

Scheme 29. Preparation of Methylpiperidine Analog CCG-211829.a

aReagents and conditions: (a) nBuLi, DIPA, THF, -78 °C; then MeI, THF, -78 °C; (b) 4

M HCl, dioxane, RT, 1 h; (c) 4a, EDC∙HCl, HOBT, TEA, DCM, RT; (d) 27, EDC∙HCl,

HOBT, TEA, DCM, RT.

The key cyclopropane of the exo-bicyclic analog CCG-222980 was installed by a

method previously reported.150, 151

An electron-deficient carbenoid was generated from

ethyl diazoacetate and treated with Cbz-protected 2,5-dihydropyrrole 111 over several

days. This yielded a mixture of unreacted starting material and two diastereomers of the

product 112, enriched in the exo-isomer, which was taken forward after tedious

separation and deprotected via hydrogenolysis and coupled with the indole carboxylic

84

acid 4a. Treatment with KOTMS hydrolyzed the ester to the potassium salt, which was

subsequently coupled with amine 27 to yield the final analog (Scheme 30).

Scheme 30. Preparation of Exo Bicyclic Analog CCG-222980.a

aReagents and conditions: (a) N2CHCO2Et, Rh2(OAc)4, DCM, reflux, 72 h, 3:1 dr; (b)

Pd/C, H2, EtOH, HCl, RT; (c) 4a, EDC∙HCl, HOBT, TEA, DCM, RT; (d) KOTMS, THF,

RT; (e) 27, EDC∙HCl, HOBT, TEA, DCM, RT.

Scheme 31. Preparation of Bridged Analog CCG-224000.a

aReagents and conditions: (a) KOTMS, THF, RT, 12 h; (b) 27, EDC∙HCl, HOBT, TEA,

DCM, RT; (c) HCl, dioxane, RT; (d) 4a, EDC∙HCl, HOBT, TEA, DCM, RT.

85

The ethylene-bridged piperidine analog CCG-224000 was first constructed by

hydrolyzing the commercially available protected amino ester 114 and coupling the

resulting acid with amine 27 to provide the latent right-hand half 115 of the analog.

Deprotection under acidic conditions followed by amide coupling with the indole

carboxylic acid 4a provided the desired analog (Scheme 31).

Scheme 32. Preparation of the Spiro Analog CCG-224220.a

aReagents and conditions: (a) PhCHO, tol, reflux, 2 h, 85%; (b) diethyl malonate, NaH,

DMF, reflux, 12 h, 84%; (c) Pd/C, H2, MeOH, RT, 6 d, 98%; (d) MsCl, TEA, DCM, RT,

3 h; (e) TsNH2, K2CO3, DMSO, 90 °C, 5 h, 48% (2 steps); (f) NaOH, H2O, EtOH, reflux,

2 h, 91%; (g) pyridine, reflux, 12 h, 92%; (h) 4-pyridylethylamine, EDCHCl, HOBT,

TEA, DCM, RT, 12 h, 80%; (i) Na0, naphthalene, THF, RT, 5 min; (j) 4a, EDCHCl,

HOBT, TEA, DCM, RT, 14 h, 61% (2 steps).

The spiro core of CCG-224220 was assembled in manner similar to that reported

(Scheme 32).152, 153

The diol 117 was protected as an acetal,154

and the bromides were

86

doubly displaced by diethylmalonate under basic conditions to give the diester 119. The

acetal was removed to reveal the alcohols of 120, which were subsequently mesylated

and displaced by tosylamide to give the cyclized spiro product 121. Ester hydrolysis and

thermal decarboxylation furnished the mono-carboxylic acid 122 in good yield, and this

was coupled with amine 27 to give the latent right-hand half 123 of the desired final

compound. Cleavage of the tosyl proved to be a substantial challenge, however. Various

conditions with samarium (II) iodide either failed to remove the tosyl, or reduced and

cleaved the amide preferentially.155, 156

Other reported methods of tosyl deprotection were

employed with no success157, 158

until sodium napthalenide was used,159

which quickly

and cleanly cleaved the S-N bond to reveal the amine 124, which was then coupled with

the indole carboxylic acid 4a to afford the desired analog CCG-224220.

Scheme 33. Preparation of Azetidine-Containing CCG-224002.a

aReagents and conditions: (a) methyl 4-azetidinecarboxylate hydrochloride, EDC, HOBT,

DIPEA, DCM, rt; (b) 10% aq. NaOH, EtOH; (c) 27, EDC, HOBT, TEA, DCM, rt.

87

The azetidine-containing analog CCG-224002 (Scheme 33)

Scheme 33was assembled in a manner similar to that used for construction of the

azetidine-containing pyrrole analogs. The indole carboxylic acid 4a was coupled with the

azetidine methyl ester, and the intermediate 125 was saponified and coupled with 4-(2-

aminoethyl)pyridine to give the final analog.

Scheme 34. Preparation of Azapane Analog CCG-222661.a

aReagents and conditions: (a) N2CHCO2Et, BF3OEt2, Et2O, -78 °CRT, 1h; (b) NaBH4,

EtOH, 0 °CRT, 2h; (c) MsCl, TEA, DCM, 0 °CRT, 6 h; (d) DBU, THF, 80 °C, 1 h; (e)

Pd/C, 50 psi H2, EtOH, HCl, RT, 24 h; (f) ) 4a, EDCHCl, HOBT, TEA, DCM, RT, 16 h;

(g) 10% aq. NaOH, EtOH; (h) 27, EDCHCl, HOBT, TEA, DCM, RT.

The core azapane of CCG-222661 was first accessed by way of ring expansion of

N-protected 4-piperidone 126 with ethyl diazoacetate and the strong Lewis acid BF3

(Scheme 34).160

The resulting racemic γ-ketoester 127 was reduced with NaBH4 to the γ-

88

hydroxy ester and mesylated. The mesylate 129 was then easily eliminated under mild

heating with DBU, and the ene ester 130 was exposed to heterogenous reduction

conditions to both reduce the alkene and the Cbz protecting group. The product amine

131 was then coupled with indole carboxylic acid 4a, saponified, and then coupled with

amine 27 to give the azapane analog CCG-222661.

Conclusion

The primary goal of this chapter was the identification of the unknown inhibitor

pharmacophore. To achieve this, conformationally-restricted analogs were prepared and

evaluated in the WEEV replicon assay. While the majority of compounds possessed

substantially reduced potency, the data pointed towards the importance of the distal

amide carbonyl to activity and a potential equatorial-equatorial relationship between the

1,4-substituents of the core piperidine ring. Intriguingly, the azapane CCG-222661 was

identified as our most potent compound to-date, with an IC50 = 0.49 ± 0.09 for the

racemate, and with an expected IC50 up to two-fold better for the active enantiomer. It is

hoped this data may be useful for the generation of a computational 3-D pharmacophore

model that may be applied to the identification of new alphavirus inhibitor templates via

in silico screening methods.

89

Metabolic Stability Chapter V.

Rationale

Metabolism is the enzymatic modification of compounds so that an organism may

enhance clearance, and is a crucial detoxification mechanism for removing reactive or

toxic xenobiotics.161

Unfortunately, even drugs considered safe may undergo this process.

Metabolism dictates the overall biological availability of a compound, and those

compounds that undergo rapid metabolism may be depleted before reaching their

intended biological target. Metabolic stability is one of the most crucial aspects of early

drug development, and instability is a potential killer of lead series.81

The majority of metabolism occurs in the liver, where a battery of metabolic

enzymes resides, although metabolism may occur elsewhere as well (blood, epithelial

intestinal cells, etc.)162, 163

These enzymes largely consist of a class called the cytochrome

p450s, or CYPs, that are responsible for Phase I metabolism, whose purpose is the

installation of polar functional groups to improve aqueous solubility to enhance clearance

and to provide handles for further Phase II metabolic pathways.79, 164

Potential sites of metabolism can be easily predicted using computational models

like that shown in Figure 19.165-169

However, while likely, empirical methods are

required to confirm such predictions because properties like atom accessibility and

overall lipophilicity are important factors. To this end, it was necessary for us to develop

90

an in-house metabolic stability assay to facilitate our ability to rapidly identify labile

functionalities and design them out of future analogs.

Figure 19. Predicted Sites of Metabolism in CCG-205432, Rank Ordered.

Predicted sites of metabolism are rank ordered where 1 = most likely, 2 = next most

likely, etc. Predictions calculated using smartCYP v.2.4.2.165, 166

The assay utilized commercially-available mouse liver microsomes harvested

from Balb/C mice, though the line of mice used by our collaborators in the lab of Prof.

David Irani for in vivo testing were C57 BL/6, and we found that the results were

translatable. Compounds were incubated in the presence of microsomes over 15 minutes,

and aliquots were removed and quenched over this time at specific time points. We also

developed an LC/MS/MS method to maximize throughput and detection sensitivity so

that parent compound concentration could be quickly assessed at concentrations as low as

single-digit nanomolar, or less than one percent of starting incubation concentration.

SAR

As discussed in Chapter I, avoidance of predicted metabolic instability led to the

replacement of the early thienopyrrole scaffold with indole. However, it remained

91

necessary to demonstrate empirically that we had gained stability through this

substitution, so we established a mouse liver microsome-based metabolic stability assay

and a corresponding LC/MS/MS analytical method for our lab.

Table 15. MLM Metabolic Stabilities for Thienopyrrole and Indole Leads.

CCG

No. Structure

IC50

(µM)a

CC50

(µM)b

MLM

T1/2

(min)c

ClogP

203881

11.1 ± 1.3 >100 1.7 ± 1.7 4.4

102516†

15.6 ± 1.8 >100 7.3 ± 3.9 4.5

203926†

6.8 ± 1.0 >100 31 ± 7.1 4.9



by the MTT reduction assay. Values are mean of at least n=3 independent experiments. cHalf-life in BALB/c mouse liver microsome incubation. Values are half-lives calculated

from the equation T1/2=ln(2)/slope, where the slope was ln(% detected) against time,

plotted from the mean of at least n=3 independent experiments. Values are mean ± SEM

of n = 3 independent experiments. †Synthesized by Janice Sindac.

Our newly developed protocol allowed for convenient in-house metabolic

stability analysis and was able to differentiate the short half-lives of our leads (Table 15).

Key to this was optimizing the concentration of microsomes, as the compounds were so

92

unstable as to be not differentiable under the conditions originally employed. Ultimately,

we found that quartering the amount relative to established protocols worked well. In the

assay, we discovered that the indole CCG-102516 had a half-life of 7.3 ± 3.9 minutes,

whereas the thienopyrrole CCG-203881 had a half-life of only 1.7 ± 1.7 minutes,

supporting our original stability hypothesis and validating our thienopyrrole/indole

switch. Despite this, CCG-102516 was still quite metabolically unstable. Interestingly,

CCG-203926, which differs by only a methyl group from CCG-102516, had

dramatically improved stability, with a half-life of 31 ± 7.1 minutes. This was

presumably due to steric hindrance of the potentially labile benzylic site.

Table 16 shows the activities of the lead indole compound CCG-205432, its

pyrrole-substituted analog CCG-206381, and the best compound from Table 15,

CCG-203926. Note that the stability of CCG-203926 (2.3 ± 0.1 minutes) does not match

its stability in Table 15 (31 ± 7.1 minutes); this was mostly likely due to the fresher,

more active microsomes used in the former study. However, the relative stabilities are not

affected, and we can confidently conclude that CCG-206381 (3.7 ± 1.3 minutes) and

CCG-205432 (16.7 ± 1.7 minutes) are substantially more stable than CCG-203926 in the

assay. This information suggests that the right-hand benzyl functionality may be a major

site of metabolism, since stability is significantly improved by replacement of the benzyl

amide with pyridylethyl substitution, although overall reduction in ClogP might also be a

factor. The data also indicate that pyrroles are far more metabolically labile than indoles

in this template. Detail regarding the application of these data appears in Chapter III.

93

Table 16. MLM Metabolic Stabilities For Indole and Pyrrole Analogs.

CCG

No. Structure

IC50

(µM)a

CC50

(µM)b

MLM T1/2

(min)c

ClogP

205432†

0.53 ± 0.1 65 16.7 ± 1.7 3.1

206381

0.68 ± 0.04 >100 3.7 ± 1.3 1.7

203926†

6.8 ± 1.0 >100 2.3 ± 0.1 4.9



by the MTT reduction assay. Values are mean of at least n=3 independent experiments. cHalf-life in BALB/c mouse liver microsome incubation. Values are half-lives calculated

from the equation T1/2=ln(2)/slope, where the slope was ln(% detected) against time,

plotted from the mean of at least n=3 independent experiments. Values are mean ± SEM

of n = 3 independent experiments. †Synthesized by Janice Sindac.

With the metabolic stability knowledge gained from Table 15 and Table 16, a

variety of proposed metabolically stable compounds were prepared and evaluated.

Pyridine CCG-212090 was over ten-fold more stable towards metabolism than CCG-

203926 (Table 17). ClogP is highly correlated with CYP metabolism,170, 171

thus CCG-

212090’s stability may be attributed to its ten-fold lower ClogP relative to CCG-203926.

Alternatively, this could suggest that the benzyl ring is a potential site of metabolism, as

pyridines are known to be less susceptible due to their electron-deficiency and

94

polarity.172, 173

While imidazoles are expected to be more stable than pyrroles by virtue of

the electron-withdrawing property of the azine nitrogen, the improved half-life of CCG-

206582 is more likely indicative of CYP inhibition, as the imidazole moiety is known to

bind to heme iron, a crucial component of CYP enzyme function.174-176

Table 17. MLM Stability and WEEV Replicon Data for Select Analogs.


b

MLM T1/2

(min)c

ClogP

205432†

0.53 ± 0.1 65 11.5 ± 0.7 3.1

203926†

6.8 ± 1.0 >100 1.8 ± 0.5 4.9

206582†

4.9 ± 2.6 >100 17.0 ± 2.7 2.7

212090†

0.6 ± 0.3 92 23.4 ± 4.0 3.4



by the MTT reduction assay. Values are mean of at least n=3 independent experiments. cHalf-life in BALB/c mouse liver microsome incubation. Values are half-lives

calculated from the equation T1/2=ln(2)/slope, where the slope was ln(% detected)

against time, plotted from the mean of at least n=2 independent experiments. Values are

mean ± SEM of n = 2 independent experiments. †Synthesized by Janice Sindac.

95

Table 18. MLM Stability and WEEV Replicon Data for Select Analogs.


b

MLM T1/2

(min)c

ClogP

205432†

0.53 ± 0.1 65 11.5 ± 0.7 3.1

206381

0.68 ± 0.04 >100 2.9 ± 0.1 1.7

211751

4.4 ± 0.5 >100 9.1 ± 3.0 1.9

209023 †

1.6 ± 0.5 46 77.9 ± 20.4 2.4

206565†

4.1 ± 0.1 75 43.0 ± 1.5 2.3



by the MTT reduction assay. Values are mean of at least n=3 independent experiments. cHalf-life in BALB/c mouse liver microsome incubation. Values are half-lives

calculated from the equation T1/2=ln(2)/slope, where the slope was ln(% detected)

against time, plotted from the mean of at least n=2 independent experiments. Values

are mean ± SEM of n = 2 independent experiments. †Synthesized by Janice Sindac.

Azaindole CCG-211751 was prepared in an effort to silence possible sites of

metabolism in the indole phenyl ring, but its lack of improved stability indicates that the

phenyl portion of the indole may only be a minor site of metabolism (Table 18).

96

Conversely, benzimidazole CCG-206565 was markedly more stable than the

corresponding indole CCG-205432, due either to stabilization of the pyrrole ring, or

simply reduced ClogP. Intriguingly, replacement of chlorine with hydrogen upon the N-

benzyl substituent of the template (CCG-209023) resulted in a dramatic half-life

improvement. This may simply be the result of lower ClogP, as compounds with high

logP are known to be susceptible to CYP450 enzymes. Furthermore, it suggests that

metabolism of the indole N-benzyl is not a major pathway. Despite having less activity

than the lead CCG-205432, this information allowed us to move CCG-209023 into

mouse studies, on the basis that enhanced metabolic stability would translate into

improved bioavailability. Gratifyingly, a ten mouse preliminary study demonstrated a

40% survival rate when infected mice were treated with the compound, marking CCG-

209023 as our most efficacious compound to-date.

Several fluorinated analogs (fluoropyrroles CCG-209020 and CCG-209021, and

Janice Sindac’s fluoroindoles CCG-208918 and CCG-208919) were synthesized to

probe potential metabolic soft spots around the pyrrole and indole rings (Table 19).

Incorporation of fluorine into compounds is a well-known and powerful tactic for

improving stability towards Phase I metabolism as fluorine is similar in size to hydrogen

(classical isostere) but is less reactive due to the strength of the C-F bond.177-179

Interestingly, these analogs, while active in the WEEV replicon assay, were also

surprisingly toxic and were therefore not promoted to MLM stability studies.

97

Table 19. WEEV Replicon Data for Fluorinated Analogs.


b

205432†

0.53 ± 0.1 65

206381

0.68 ± 0.04 >100

209021

0.93 ± 0.02 8.0

208918†

0.73 ± 0.05 24

208919†

0.92 ± 0.05 52


bCell viability

determined by the MTT reduction assay. Values are mean of at least n=3 independent

experiments. †Synthesized by Janice Sindac.

98

Synthesis

Scheme 35. Preparation of 4-Fluoropyrrole Analogs.a

aReagents and conditions: (a) TEMPO, TCCA, DCM, 0 °CRT, 20 min, 99%; (b) DAST,

DCM, -78 °CRT, 12, h, 86%; (c) 4 M HCl/1,4-dioxane, THF, RT, 4 h, 100%; (d) MnO2,

THF, reflux, 3 h, 20%; (e) 4-chlorobenzyl chloride, K2CO3, DMF, 60 °C, 30 h, 83%; (f)

10% aq. NaOH, EtOH, RT, 18 h, 78%; (g) ) NH2R1, EDCHCl, HOBT, TEA, DCM, rt,

15 h, 52%.

Fluoropyrroles CCG-209020 and CCG-209021 were prepared in a manner

similar to that previously described (Scheme 35).180

The 4-hydroxyproline 133 was

oxidized to 4-ketoproline 134 under TEMPO catalysis, and this was subsequently

fluorinated with DAST to provide the germinal fluoroproline 135. After acid-mediated

deprotection, the resulting 1H-proline was aromatized to the 4-fluoropyrrole 127 with

99

MnO2.181

Alkylation with 4-chlorobenzyl choride gave the N-benzyl intermediate, which

was saponified and coupled with the appropriate amine.

Scheme 36. Preparation of 6-Azaindole Analog CCG-211751.a

aReagents and conditions: (a) TFAA, 0 °C, 2 h; then Fum. HNO3, RT, 12 h; then

Na2S2O5, H2O, RT, 24 h, 9%; (b) Na0, diethyl oxalate, EtOH, tol, RT, overnight, 40%; (c)

Pt/C, 1 ATM H2, EtOH, AcOH, RT, 24 h; (d) EtOH, K2CO3, RT, 18 h, 51% (2 steps); (e)

2a, t-BuOK, DMF, RT, 14 h, 65%; (f) 10% aq. NaOH, EtOH, RT, 24 h, 84%; (g) 36,

EDCHCl, HOBT, TEA, DCM, RT, 20 h, 55%.

Azaindole analog CCG-211751 was prepared using largely known procedures

(Scheme 36).182

First, 4-picoline was nitrated under inefficient but mild conditions as

reported by Katritzky.183

The resulting 3-nitro-4-picoline 141 was then treated with

sodium in EtOH to form the anion, which was subsequently treated with diethyloxalate to

100

give the β-ketoester 142. The nitro was then fully reduced to the amine under

heterogeneous catalysis, and, upon introduction of basic conditions, this cyclized via

addition/elimination to yield the desired 6-azaindole core 143. Alkylation with 2a was

accomplished by fully deprotonating the azole nitrogren with tBuOK before addition of

the electrophile. Weaker bases, such as alkali metal carbonates (e.g. Cs2CO3) returned

mostly pyridinium salts. Saponification of the ester 144 and amidation with the amine

dihydrochloride 36 provided the desired azaindole analog CCG-211751.

Conclusion

The primary goal of this chapter was the development of a mouse-liver

microsome assay and LC/MS/MS protocol suitable for prioritizing compounds based

upon their stability towards Phase I metabolism. To this end, we designed a relatively

high-throughput MLM method that was successful in assessing and identifying

compound stabilities, and that correlated well with in vivo pharmacokinetic data.

Moreover, it facilitated identification of important modifications (e.g. pyrrole-to-aniline

substitution and chlorine-to-hydrogen substitution) that significantly improved stability,

leading to the discovery of our most stable compound to-date (CCG-209023).

101

Future Directions Chapter VI.

Overall, our design and development of inhibitors of neurotropic alphavirus

replication has been successful in identifying and optimizing potent compounds both in

vitro and in vivo against a broad spectrum of alphaviruses. Furthermore, we have

successfully improved physicochemical properties both predictive and demonstrable of

enhanced bioavailability and BBB-penetration. This is a significant step towards the

development of a successful antiviral for neurotropic alphaviruses, as there are no known

antivirals that possess CNS-permeability that are also therapeutically useful in the

treatment of these viruses.15, 40

Furthermore, the general mechanism of action is unique as there are few known

antivirals that target host machinery. Classically, such targets were avoided due to

assumed toxicity issues, but the low toxicity of our compounds places them firmly within

a small promising class of new host-targeting antivirals, and the only ones specific for

alphaviruses.184-187

The advantages of such a compound include enhanced broad-

spectrum activity and – more importantly – significantly reduced incidence of resistance.

Due to relatively rapid viral replication and poor fidelity, resistance-conferring mutations

in viral proteins may arise after treatment with viral-targeting drugs, which places a large

emphasis on the development of new drugs. On the other hand, due to the relatively low

turnover of host cells, resistance-conferring mutations are expected to occur much more

slowly, if at all, with host-targeting antivirals.

102

However, an important aspect of medicinal chemistry is the establishment of a

pharmacophore model – the collection of structural and electronic features requisite for

engaging with binding site contacts and maximizing activity. Importantly, such a model

facilitates the design and development of future analogs, including those based on

fundamentally different scaffolds. Over the course of the WEEV inhibitor project, we

identified multiple attributes critical to activity, such as the potency-length dependence

(Chapter II) and the disposition of the right-hand secondary amide (Chapter IV). With

the aid of our collaborator Paul Kirchhoff, we have attempted to computationally develop

a three-dimensional pharmacophore model of our compounds and apply such a model to

an in silico high-throughput screen to discover new chemical templates. Unfortunately,

we have been unable to generate a predictive pharmacophore model; models built from

diverse WEEV inhibitor training sets could not discriminate between known active and

inactive compounds. Moving forward, we would like to direct studies toward

identification of the pharmacophore through the design, synthesis, and evaluation of new

analogs.

Related to pharmacophore identification, we have also noted conformation-

dependent recognition by MDR1 in this project. Despite the magnitude of on-going work

in the field of medicinal chemistry, this observation has been noted only rarely in the

literature. Historically, tremendous efforts have been expended by medicinal chemists in

identifying structure-activity relationships for small molecule interactions with MDR1,

but the SAR is still largely undefined due to the promiscuity of the transporter. Therefore,

research regarding conformation-activity relationships could significantly enhance the

design of transporter-evasive compounds.

103

Another important aspect of drug development is the identification of the biological

target, and by extension, the mechanism of action through which the compounds exert

their pharmacological effect. Our collaborators have made tremendous strides towards

this end,104

and Janice Sindac has developed several photo-affinity probes to assist in

target identification. In contrast to the phenotype-based approach to analog design

employed throughout the project, such information could facilitate a target-based avenue

to future drug development. In this way, knowledge of the target could lead to models of

the binding site (e.g. homology) that could allow us to predict and incorporate structural

features tailored specifically to engage with potential binding site contacts. Also, because

toxicity is often a product of off-target binding to structurally similar biological

macromolecules, this could also allow us to improve the toxicity profile of our analogs,

especially in light of their host-based mechanism of action. Therefore, the continued

design and development of probes – no trivial endeavor – is an important aspect of the

project’s future.

104

Experimental Data Chapter VII.

All reagents were used as received from commercial sources unless otherwise noted. 1H

and 13

C spectra were obtained in DMSO-d6 or CDCl3 at room temperature, unless

otherwise noted, on a Varian Inova 400 MHz, Varian Inova 500 MHz, or Bruker Avance

DRX 500 instruments. Chemical shifts for the 1H NMR and

13C NMR spectra were

recorded in parts per million (ppm) on the δ scale from an internal standard of residual

tetramethylsilane (0 ppm). Rotamers are described as a ratio of rotamer A and B if

possible. Otherwise, if the rotamers cannot be distinguished, the NMR peaks are

described as multiplets. Mass spectroscopy data were obtained on a Waters Corporation

LCT. HPLC retention times were recorded in minutes (min) using an Agilent 1100 series

with an Agilent Zorbax Eclipse Plus–C18 column with the gradient 10% ACN/water (1

min), 10–90% ACN/water (6 min), and 90% ACN/water (2 min).

Reagent abbreviations and acronyms: ACAC (acetylacetone), AcOH (acetic acid), Ac2O

(acetic anhydride), AlCl3 (aluminum chloride), Ar (argon), BnBr (benzyl bromide),

Boc2O (di-tert-butyl dicarbonate), Cs2CO3 (cesium carbonate), Cu0 (elemental copper),

Cu2O (copper (I) oxide), DAST (diethylaminosulfur trifluoride), DCM

(dichloromethane), DIPA (diisopropylamine), DIPEA (N,N-diisopropylethylamine,

Hünig’s base), DMF (N,N-dimethylformamide), DPPP (1,3-

105

bis(diphenylphosphino)propane, EDCHCl (1-Ethyl-3-(3-

dimethylaminopropyl)carbodiimide hydrochloride), EGDME (ethylene glycol dimethyl

ether), Et2O (diethyl ether), EtOAc (ethyl acetate), EtOH (ethanol), H2 (hydrogen),

HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid

hexafluorophosphate), H2O (water), HBr (hydrobromic acid), HCl (hydrogen chloride or

hydrochloric acid), Hex (hexanes), HMDS (hexamethyldisilazane), HOBT (1-

hydroxybenzotriazole), ind (indane), IPA (isopropanol), K2CO3 (potassium carbonate),

KOH (potassium hydroxide), LiAlH4 (lithium aluminum hydride), LiBr (lithium

bromide), m-CPBA (meta-chloroperoxybenzoic acid), MeCN (acetonitrile), MeI (methyl

iodide), MeOH (methanol), MgCl2 (magnesium chloride), MgSO4 (magnesium sulfate),

MnO2 (manganese (IV) oxide), N2 (nitrogen), Na0 (elemental sodium), NaBH4 (sodium

borohydride), NaBH3CN (sodium cyanoborohydride), NaBH(OAc)3 (triacetoxy sodium

borohydride), Na2CO3 (sodium carbonate), Na2SO4 (sodium sulfate), NAPDH

(nicotinamide adenine dinucleotide phosphate), NaH (sodium hydride), NaHCO3 (sodium

bicarbonate), NaNO2 (sodium nitrite), NaOH (sodium hydroxide), Na2S2O5 (sodium

metabisulfite), NH3 (ammonia), n-BuLi (n-butyl lithium), Pd/C (palladium on carbon),

PhCl (chlorobenzene), PhLi (phenyllithium), PhNH2 (aniline), PhN(Tf)2 (N,N-

bis(trifluoromethylsulfonyl)anilie, PhSeCl (phenylselenyl chloride), PPh3

(triphenylphosphine), Pt/C (platinum on carbon), PtO2 (platinum (IV) oxide), pyr

(pyridine), s-BuLi (sec-butyl lithium), SmI2 (samarium (II) iodide), SnCl2 (tin (II)

chloride), t-BuOH (tert-butanol), t-BuOK (potassium tert-butoxide), TCCA

(trichloroisocyanuric acid), TEA (triethylamine), TEMPO ((2,2,6,6-tetramethylpiperidin-

1-yl)oxy), TFA (trifluoracetic acid), TFE (trifluoroethanol), TFFA (trifluoroacetic

106

anhydride), TFE (2,2,2-trifluoroethanol); THF (tetrahydrofuran), tol (toluene), TsOH

(toluenesulfonic acid), Zn0 (elemental zinc), ZnCl2 (zinc chloride).

Condition abbreviations and acronyms: ATM (atmosphere), aq. (aqueous), conc.

(concentrated), d (days), fum. (fuming), g (grams), h (hours), M (molar), mg

(milligrams), mL (milliliters), µL (microliters), mM (millimolar), min (minutes), psi

(pressure per square inch), RT (room temperature), wt. (weight).

Biology

Mouse Microsomal Metabolic Stability Procedure. Balb-C mouse liver

microsomes were purchased from Invitrogen. MgCl2 and NADPH were obtained from

Sigma. Solvents were of HPLC grade or better. The HPLC-MS/MS system consisted of a

ThermoElectron Finnigan TSQ Quantum Ultra AM.

Microsomal incubations were done in triplicate. Incubation mixtures contained 2

µL of 20mg/mL microsomes (approximately 0.04 mg microsomal protein), 4 µL of 100

µM DMSO-dissolved substrate (1.0 µM final), in 475 µL potassium phosphate buffer

(0.1 M, pH 7.5, containing 3.3 mM MgCl2.) The incubation mixture was allowed to shake

at 37 °C for 5 min, and a T=0 aliquot was removed. Reaction was initiated by the

addition of 20 µL of NaDPH (22 mM under the same buffer conditions) and the mixture

was allowed to shake at 37 °C until the final aliquot was removed. 30 µL aliquots were

taken after 0 minutes, 5 minutes, and 15 minutes. All aliquots were quenched by dilution

in 90 µL MeCN containing an internal standard and the precipitate was pelleted via

centrifugation. 20 µL of supernatant was injected onto the HPLC-MS/MS. Mobile phase

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A was 95:5 H2O:MeCN with 0.1% formic acid, and mobile phase B was MeCN with

0.1% formic acid. Column used was a Luna C18(2) 4.6 X 30 mm column with 3µM

particle size. The flow rate was 2 mL/min and a gradient mobile phase composition was

used: isocratic hold for 1 min at 90% A (10% B), 1 min gradient to 10% A (90% B), 1

min gradient back to 90% A (10% B), and 1 min isocratic hold at 90% A (10% B.)

Ionization method consisted of positive electrospray. Source parameters were optimized

for each individual substrate. Substrates and internal standards were followed by selected

reaction monitoring. Substrate/internal standard area ratios were determined and

converted into percent substrate remaining. The natural log of the percent remaining was

plotted against time, the slope of the linear regression was determined, and the equation

T1/2 = – ln(2)/k was used to calculate half-life.

Chemistry

Ethyl 1-(4-chlorobenzyl)-1H-indole-2-carboxylate (3a). Ethyl 1H-indole-2-

carboxylate 1 (4.50 g, 23.78 mmol) was dissolved in anhydrous DMF (60 mL) under N2

at RT, then cooled to -10 °C. Granular t-BuOK (2.94 g, 26.20 mmol) was added to the

solution, eliciting a slight yellow color. 4-Chlorobenzyl chloride 2a (4.02 g, 24.97 mmol)

– pre-dissolved in anhydrous DMF (5 mL) – was added via syringe pump at 0.1 mL/min

at -10 °C. After this time, was allowed to stir at RT for 12 h. The reaction was then

diluted with 1:1 EtOAc:Hex and quenched by the addition of sat. aq. NaHCO3. The

organic phase was then washed with H2O (10 X) and brine (1X), dried (MgSO4), and

concentrated to give the pure compound as a white solid that was subsequently washed

thoroughly with hexanes. Yield: 6.80 g, 21.67 mmol, 91%. 1H NMR (500 MHz,

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Chloroform-d) δ 7.78 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 1.6 Hz, 1H), 7.38 – 7.34 (m, 2H),

7.25 (ddd, J = 15.1, 8.4, 2.8 Hz, 3H), 7.07 – 7.01 (m, 2H), 5.83 (s, 2H), 4.39 (qd, J = 7.1,

1.6 Hz, 2H), 1.42 (td, J = 7.1, 1.6 Hz, 3H).

Ethyl 1-(4-fluorobenzyl)-1H-indole-2-carboxylate (3b). Ethyl 1H-indole-2-

carboxylate 1 (500 mg, 2.64 mmol) was dissolved in anhydrous DMF (8 ml), followed by

1-(chloromethyl)-4-fluorobenzene 2b (0.380 ml, 3.17 mmol) and the addition of solid

K2CO3 (730 mg, 5.29 mmol). This was stirred at 60 °C for 20 h, at which time the

reaction was allowed to cool to room temperature. Precipitate formed upon the addition

of water (40 mL), was isolated over a filter, and dried under high vacuum overnight to

give 550 mg of the title compound as a white solid which was recrystallized from Et2O.

Yield: 550 mg, 1.850 mmol, 70%. 1H NMR (500 MHz, Chloroform-d) δ 7.72 (d, J = 7.9

Hz, 1H), 7.40 (s, 1H), 7.37 – 7.31 (m, 2H), 7.18 (ddd, J = 7.7, 6.5, 1.3 Hz, 1H), 7.05 (dd,

J = 8.4, 5.3 Hz, 2H), 6.94 (t, J = 8.5 Hz, 2H), 5.81 (s, 2H), 4.34 (q, J = 7.1 Hz, 2H), 1.38

(t, J = 7.1 Hz, 3H).

1-(4-Chlorobenzyl)-1H-indole-2-carboxylic acid (4a). Ethyl 1-(4-chlorobenzyl)-

1H-indole-2-carboxylate 3a (5.95 g, 18.96 mmol) was added to a mixture of 10% aq.

NaOH (13.7 mL, 37.9 mmol), EtOH (15 mL), and THF (15 mL), but did not completely

dissolve. This suspension stirred at RT for 14 h, over which time all material dissolved

and the reaction proceeded to completion. Solvent was stripped off in vacuo, the residue

was taken up in H2O and acidified to pH<1 with conc. HCl at 0 °C. The resulting

precipitate was collected over a filter and washed with cold 1 M HCl, air-dried, then

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dried thoroughly under high vacuum to give a white powder. No further purification

necessary. Yield: 5.20 g, 18.20 mmol, 96%. 1H NMR (500 MHz, Chloroform-d) δ 7.76

(d, J = 8.1 Hz, 1H), 7.56 (s, 1H), 7.40 – 7.31 (m, 2H), 7.27 – 7.16 (m, 3H), 7.00 (d, J =

8.2 Hz, 2H), 5.81 (s, 2H).

1-(4-Fluorobenzyl)-1H-indole-2-carboxylic acid (4b). Ethyl 1-(4-fluorobenzyl)-

1H-indole-2-carboxylate 3b (409 mg, 1.376 mmol) was suspended in ethanol (5 mL) and

7M NaOH (5 mL) and stirred at 50 °C for 3 h. Ethanol content was reduced in vacuo.

The solution was then acidified to pH 2 and the precipitate was collected over a filter,

washed with a small amount of ice cold-water, air dried on the filter, and then finally

vacuum dried to give the title compound as a fine, white powder. Yield: 360 mg, 1.337

mmol, 97%. 1H NMR (500 MHz, Chloroform-d) δ 8.41 (d, J = 5.8 Hz, 1H), 7.81 – 7.71

(m, 4H), 7.70 – 7.60 (m, 3H), 7.18 (d, J = 5.7 Hz, 1H), 5.90 (d, J = 5.5 Hz, 2H).

Tert-Butyl 4-(hydroxymethyl)piperidine-1-carboxylate (7). Piperidine-4-methanol

6 (3.74 g, 32.5 mmol) and Boc2O (8.5 g, 38.9 mmol) were added to THF (50 mL) at RT,

followed by Na2CO3 (6.88 g, 64.9 mmol) and H2O (30 mL). Addition was mildly

exothermic. The reaction was refluxed at 100 °C for 2 h, after which it was allowed to

cool to RT. The reaction was then extracted with EtOAc, and the extract was washed

with brine (1X), dried (MgSO4), and concentrated. The residue was purified by silica

flash chromatography (150 g silica, 50% EtOAc:Hex). Concentration provided a white

solid. Yield: 6.09 g, 28.3 mmol, 87%. 1H NMR (500 MHz, Chloroform-d) δ 4.10 (s, 2H),

110

3.47 (t, J = 5.2 Hz, 1H), 2.69 (s, 2H), 1.70 (d, J = 13.2 Hz, 2H), 1.62 (ddp, J = 15.2, 6.7,

4.0 Hz, 1H), 1.44 (s, 9H), 1.12 (qd, J = 12.5, 4.4 Hz, 2H).

Tert-Butyl 4-formylpiperidine-1-carboxylate (8). Tert-butyl 4-

(hydroxymethyl)piperidine-1-carboxylate 7 (1.50 g, 6.97 mmol) and anhydrous DMSO

(1.24 mL, 17.42 mmol) were dissolved in DCM (100 mL) at RT, then cooled to -78 °C.

(COCl)2 (1.22 mL, 13.93 mmol) was then added dropwise under N2 and the reaction was

allowed to stir for 30 min at -78 °C. Still at this temperature, TEA (4.86 mL, 34.8 mmol)

was added dropwise and a slight yellow color change was observed. The reaction was

allowed to stir for 30 min at -78 °C, then 3 h at 0 °C. After this time, the reaction was

quenched by the addition of 0.1 M HCl and material was extracted with EtOAc. The

extract was washed with 0.1 M HCl (2X), H2O (3X), and brine (1X), then dried (MgSO4)

and concentrated in vacuo. This was immediately purified via silica flash

chromatography (100 g silica, 40% EtOAc:Hex) to provide the desired product as a white

crystal after removal of solvent. Yield: 1.32 g, 6.19 mmol, 89%. 1H NMR (400 MHz,

Chloroform-d) δ 9.58 (s, 1H), 3.99 – 3.82 (m, 2H), 2.85 (t, J = 11.9 Hz, 2H), 2.35 (dt, J =

10.5, 6.0 Hz, 1H), 1.82 (d, J = 12.4 Hz, 2H), 1.52 – 1.44 (m, 2H), 1.37 (s, 9H).

Tert-Butyl 4-(piperidin-1-ylmethyl)piperidine-1-carboxylate (9). Piperidine (1.2

mL, 11.73 mmol) was dissolved in TFE (10 mL) over 3 Å MS, then a drop of glacial

AcOH (~ 50 µL) was added. Tert-butyl 4-formylpiperidine-1-carboxylate 8 (500 mg,

2.35 mmol) was added slowly with stirring at RT. NaCNBH3 (192 mg, 4.69 mmol) was

added and the reaction was stirred at RT for 19 h. After this time, the reaction was diluted

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with EtOAc and washed with 10% aq. Na2CO3 (3X), dried (MgSO4), concentrated under

high vacuum. The residue was then purified by silica flash chromatography (60 g silica,

70% to 100% EtOAc:Hex). Yield: 477 mg, 1.69 mmol, 72%. 1H NMR (500 MHz,

Chloroform-d) δ 4.03 (bs, 2H), 2.64 (bs, 2H), 2.28 (bs, 3H), 2.08 (d, J = 6.4 Hz, 2H),

1.71 – 1.45 (m, 10H), 1.42 (s, 9H), 1.03 (m, 2H).

Tert-Butyl 4-(cyclohexyl(hydroxy)methyl)piperidine-1-carboxylate (10). Tert-

butyl 4-formylpiperidine-1-carboxylate 8 (250 mg, 1.172 mmol) was dissolved in

anhydrous Et2O (20 mL) and a 2 M solution of cyclohexylmagnesium chloride (0.62 mL,

1.23 mmol) in Et2O was added dropwise to this under N2. The reaction was allowed to

stir at RT for 2 h, after which time the reaction was quenched by the addition of EtOH

then sat. aq. NaHCO3. Material was extracted with EtOAc, and the extract was dried

(MgSO4) and concentrated to give the desired alcohol as a solid, which was used directly

in the subsequent reaction without purification. Yield: 296 mg, 0.20 mmol, 85%.

Tert-Butyl 4-(dibenzylamino)piperidine-1-carboxylate (13). Tert-butyl 4-

aminopiperidine-1-carboxylate 12 (200 mg, 1.00 mmol) was dissolved in DCM (3 mL),

followed by the addition of H2O (1.5 mL) and granular Na2CO3 (318 mg, 3.00 mmol),

and finally benzyl bromide (0.30 mL, 2.50 mmol). The reaction was stirred at reflux (55

°C) for 3 h, after which H2O (10 mL) was added and the material was extracted with

EtOAc. The extract was dried (MgSO4) and concentrated, and the resulting residue was

purified by silica flash chromatography (20 g silica, 5% EtOAc:Hex) to give the desired

amine as a white solid. Yield: 330 mg, 0.87 mmol, 87%. 1H NMR (500 MHz,

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Chloroform-d) δ 7.40 (d, J = 7.2 Hz, 4H), 7.33 (t, J = 8.2, Hz, 4H), 7.29 – 7.23 (m, 2H),

4.18 (s, 2H), 3.67 (s, 4H), 2.69 (tt, J = 11.8, 3.5 Hz, 1H), 2.59 (bs, 2H), 1.84 (d, J = 12.4

Hz, 2H), 1.63 – 1.52 (m, 2H), 1.52 – 1.46 (m, 9H).

4-(3-Bromopropyl)pyridine (16). 3-(Pyridin-4-yl)propan-1-ol (500 mg, 3.64

mmol) was dissolved in conc. HBr (1 mL) in a pressure vessel stirred at 130 °C for 2h,

after which the reaction was allowed to cool. The reaction was then basified to pH>14

with 10% aq. NaOH at 0 °C and extracted with EtOAc (3X). The combined extracts were

dried (MgSO4) and concentrated. The residue was then purified on a silica plug (20 g

silica, 100% EtOAc) to give a crude oil that was taken into the next reaction without

further purification. Yield: 292 mg, 1.46 mmol, 40%. ). TOF ES+ MS: (M + H, Br79

)

200.0, (M + H, Br81

) 202.0.

Tert-Butyl 4-(3-(pyridin-4-yl)propyl)piperazine-1-carboxylate (17). t-Butyl

piperazine-1-carboxylate (186 mg, 1.00 mmol) was dissolved in anhydrous DMF (10

mL) under N2, and a 60% oil suspension of NaH (120 mg, 3.00 mmol) was added. After

stirring at RT under N2 for 1 h, 4-(3-bromopropyl)pyridine (200 mg, 1.00 mmol) was

added and the reaction was allowed to stir for 19 h. After this time, the reaction was

quenched with sat. aq. NaHCO3 then diluted with EtOAc and washed with H2O (3X),

10% aq. Na2CO3 (3X), and brine (1X), then dried (MgSO4) and concentrated. The residue

was purified by silica flash chromatography (25 g silica, 1% to 10% methanolic

ammonia:EtOAc). Yield: 201 mg, 0.660 mmol, 66%. 1H NMR (500 MHz, Chloroform-d)

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δ 8.49 (d, J = 5.9 Hz, 2H), 7.11 (d, J = 5.8 Hz, 2H), 3.45 – 3.39 (m, 4H), 2.65 (t, J = 7.7

Hz, 2H), 2.42 – 2.31 (m, 6H), 1.83 (p, J = 7.6 Hz, 2H), 1.46 (s, 9H).

(1-(4-Fluorobenzyl)-1H-indol-2-yl)(4-(hydroxymethyl)piperidin-1-yl)methanone

(CCG-206462). The following was added sequentially to DMF (10 mL): 1-(4-

fluorobenzyl)-1H-indole-2-carboxylic acid (1.175 g, 4.36 mmol), DIPEA (2.29 mL,

13.09 mmol), EDCHCl (1.00 g, 5.24 mmol), and HOBT (802 mg, 5.24 mmol). This

solution was allowed to stir at RT for 1.5 h, at which time 4-piperidinemethanol (754 mg,

6.55 mmol) was added. Stirring continued for 22 h at RT, at which time the solution was

partitioned between H2O and 1:1 solution of EtOAc:Et2O. The organic extract was then

washed with saturated aq. Na2CO3, dried with MgSO4, and concentrated in vacuo.

Purification was accomplished via silica flash chromatography (100 g silica, 80%

EtOAc/Hexanes.) Resulting residue was crystallized via Et2O:Hex trituration to give the

title compound as a white solid. Yield: 1.51 g, 4.11 mmol, 94%. 1H NMR (400 MHz,


7.14 (t, J = 7.5 Hz, 1H), 7.10 – 7.04 (m, 2H), 6.91 (t, J = 8.2 Hz, 2H), 6.60 (s, 1H), 5.46

(s, 2H), 4.64 (bs, 1H), 4.14 (bs, 1H), 3.42 (t, J = 5.1 Hz, 2H), 2.76 (s, 2H), 1.44 (t, J = 5.0

Hz, 1H), 1.05 (bs, 1H), 0.71 (bs, 1H).

1-(1-(4-Fluorobenzyl)-1H-indole-2-carbonyl)piperidine-4-carbaldehyde (20). (1-

(4-fluorobenzyl)-1H-indol-2-yl)(4-(hydroxymethyl)piperidin-1-yl)methanone (762 mg,

2.08 mmol) was dissolved in DCM (80 mL) in dry glassware under N2. The solution was

chilled to -78 °C and anhydrous DMSO (0.37 mL, 5.20 mmol) and (COCl)2 (0.36 mL,

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4.16 mmol) were added. The reaction was allowed to stir for 30 min, after which TEA

(1.45 mL, 10.40 mmol) was added dropwise and stirring continued for 1 h, after which

the temperature was elevated to 0 °C and stirring continued for 30 min. At this time, the

solution was taken up in EtOAc while still cold and immediately washed with 0.1 M HCl

(2X), dried (MgSO4), and concentrated to give a white solid. Yield: 527 mg, 1.46 mmol,

70%. 1H NMR (500 MHz, Chloroform-d) δ 9.61 (s, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.40

(d, J = 8.2 Hz, 1H), 7.32 – 7.26 (m, 1H), 7.19 – 7.14 (m, 1H), 7.12 – 7.06 (m, 2H), 6.93

(t, J = 8.6 Hz, 2H), 6.63 (s, 1H), 5.48 (s, 2H), 4.55 (bs, 1H), 4.07 (s, 1H), 2.50 – 2.41 (m,

1H), 2.16 – 1.93 (m, 2H), 1.84 – 1.47 (m, 4H).

Methyl 1-(4-chlorobenzyl)-1H-pyrrole-2-carboxylate (23a). Methyl 1H-pyrrole-2-

carboxylate (2.77 g, 22.14 mmol) was dissolved in anhydrous DMF (15 mL). Granular

K2CO3 (1.94 g, 26.6 mmol) was added, followed by the addition of 4-chlorobenzyl

chloride (1.80 mL, 26.6 mmol). The reaction was permitted to stir at 60 °C for 24 h, then

again 90 °C for 48 h. Cs2CO3 (21.6 g, 66.4 mmol) was then added, and the reaction was

allowed to stir at 90 °C for 24 h, then NaI (332 mg, 2.214 mmol) was added and stirring

continued at 90 °C for 24 h. At this time, the reaction was allowed to cool to RT, diluted

with EtOAc, and washed with brine (3×). The organic phase was then dried (MgSO4),

concentrated in vacuo, and purified by silica flash chromatography (150 g silica, 5%

EtOAc:Hex) to provide methyl 1-(4-chlorobenzyl)-1H-pyrrole-2-carboxylate as colorless

crystals. Yield: 4.91 g (89%). 1H NMR (400 MHz, CDCl3) δ 7.32 (d, J = 4.7 Hz, 1H),

7.26 (d, J = 3.5 Hz, 1H), 7.05–6.98 (m, 3H), 6.92–6.86 (m, 1H), 6.20 (dd, J = 3.9, 2.6 Hz,

1H), 5.52 (s, 2H), 3.76 (s, 3H).

115

Ethyl 1-(4-chlorobenzyl)-1H-imidazole-2-carboxylate (23b). Ethyl 1H-imidazole-

2-carboxylate (4 g, 28.5 mmol), 4-chlorobenzyl chloride (4.38 mL, 34.3 mmol), and

Na2CO3 (3.63 g, 34.3 mmol), was dissolved in anhydrous DMF (8 mL). The solution was

stirred at RT for 24 h, at which time H2O was added and material was extracted with

EtOAc. The organic phase was collected, dried over MgSO4, and decanted. Purification

accomplished via silica flash chromatography (150 g silica, 10% EtOAc/Hexanes to 80%

EtOAc/Hex.) The title compound was obtained as a clear, yellow-tinted oil. Yield: 7.28 g,

27.5 mmol, 96%. 1H NMR (400 MHz, CDCl3) δ 7.29 (d, J = 8.4 Hz, 2H), 7.18 (d, J = 0.9

Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 0.9 Hz, 1H), 5.59 (s, 2H), 4.37 (q, J = 7.2

Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H).

Ethyl 1-(1-(4-chlorobenzyl)-1H-pyrrole-2-carbonyl)piperidine-4-carboxylate

(24a). To methyl 1-(4-chlorobenzyl)-1H-pyrrole-2-carboxylate (4.5 g, 18.02 mmol) was

added to EtOH (50 mL) and 10% aq. NaOH (20 mL) and stirred overnight at RT, then

again overnight at 50 °C to give completion. The EtOH was stripped by rotary

evaporation until material began to precipitate, at which point the aqueous mixture was

cooled in an ice bath and acidified with conc. HCl, which elicited further precipitation.

The precipitate was collected over a filter and washed with a small amount of cold 1 M

HCl and Hex. The material was then dried under high vacuum to provide the carboxylic

acid as a white powder. Yield: 4.20 g, (99%). 1H NMR (400 MHz, DMSO-d6) δ 7.34

(d, J = 8.2 Hz, 2H), 7.19 (s, 1H), 7.06 (d, J = 8.2 Hz, 2H), 6.82 (s, 1H), 6.16–6.08 (m,

1H), 5.53 (s, 2H).

116

The following were added sequentially to anhydrous DMF (20 mL), 1-(4-

chlorobenzyl)-1H-pyrrole-2-carboxylic acid (2.0 g, 8.5 mmol), DIEA (4.45 mL, 25.5

mmol), EDCI (1.79 g, 9.34 mmol), and HOBt (1.43 g, 9.34 mmol), and this solution was

allowed to stir at RT for 30 min. Finally, ethyl isonipecotate (1.44 mL, 9.34 mmol) was

added and the reaction was permitted to stir at RT for 24 h. At this time, DCM was

removed in vacuo and the residue was dissolved in ethyl acetate, which was subsequently

washed with 1 M HCl (3X), H2O (2X), 10% aq. Na2CO3 (2X), and at last brine (1X). The

organic layer was then dried (MgSO4) and concentrated in vacuo. The residue was

crystallized from EtOAc:Hex via the slow evaporation of solvent at RT to give ethyl 1-

(1-(4-chlorobenzyl)-1H-pyrrole-2-carbonyl)piperidine-4-carboxylate as large translucent

crystals. Yield: 2.16 g (68%). 1HNMR (400 MHz, CDCl3) δ 7.24 (d, J = 8.4 Hz, 2H),

7.03 (d, J = 8.4 Hz, 2H), 6.84−6.74 (m, 1H), 6.31 (dd, J = 3.7, 1.5 Hz, 1H), 6.18−6.06 (m,

1H), 5.27 (s, 2H), 4.21 (d, J = 11.5 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 2.94 (t, J = 11.5 Hz,

2H), 2.46 (tt, J = 10.7, 4.0 Hz, 1H), 1.79 (d, J = 12.7 Hz, 2H), 1.56−1.30 (m, 2H), 1.25 (t,

J = 7.1 Hz, 3H).

1-(1-(4-Fluorobenzyl)-1H-pyrrole-2-carbonyl)piperidine-4-carboxylic acid (25a).

Ethyl 1-(1-(4-chlorobenzyl)-1H-pyrrole-2-carbonyl)piperidine-4-carboxylate (700 mg,

1.867 mmol) was dissolved in EtOH (60 mL) and H2O (20 mL). Granular LiOH (134 mg,

5.60 mmol) was added to this, and the reaction was allowed to stir at RT for 24 h. After

this time, most of the solvent was removed in vacuo, and the remaining aqueous solution

was chilled to 0 °C and acidified by concentrated HCl, which elicited a thick, sticky

precipitate that adhered to the flask. The aqueous solution was decanted, and a small

117

amount of 1 N HCl was added, and the material sonicated. The aqueous solution was

again decanted, and a small amount of ice-cold water was added, mixed, and decanted.

Remaining water and HCl was then removed by rotary evaporation, followed by high-

vacuum drying to afford the title compound as a white powder. Yield: 620 mg (96%).

1HNMR (400 MHz, DMSO-d6) δ 7.33 (dd, J = 9.0, 2.7 Hz, 2H), 7.14−6.97 (m, 2H), 6.27

(dd, J = 3.6, 1.5 Hz, 1H), 6.10−5.94 (m, 1H), 5.25 (s, 2H), 3.98 (d, J = 11.0 Hz, 2H),

3.03−2.80 (m, 2H), 2.35−2.19 (m, 1H), 1.64 (d, J = 11.1 Hz, 2H), 1.24 (d, J = 11.5 Hz,

2H).

Methyl 1-(1-(4-chlorobenzyl)-1H-pyrrole-2-carbonyl)azetidine-3-carboxylate

(29). 1-(4-chlorobenzyl)-1H-pyrrole-2-carboxylic acid (500 mg, 2.12 mmol) was added

to DCM (20 mL), followed sequentially by DIPEA (1.1 mL, 6.36 mmol), EDCHCl (447

mg, 2.33 mmol), HOBT (357 mg, 2.33 mmol), and methyl azetidine-3-carboxylate

hydrochloride (387 mg, 2.55 mmol). The reaction was allowed to stir at rt for 18 h, at

which time the DCM was removed in vacuo and the residue was taken up in 2:1

EtOAc:Et2O and washed with 1 M aq. HCl (5X), H2O (5X), 10% aq. Na2CO3 (5X), and

brine (1X), then dried (MgSO4) and concentrated. The resulting residue was purified by

silica flash chromatography (25 g silica, 5% to 30% EtOAc/Hex) to give the desired

compound as a clear, slightly yellow oil. Yield: 508 mg, 1.53 mmol, 72%. 1H NMR (500

MHz, Chloroform-d) δ 7.25 (d, J = 8.3 Hz, 2H), 7.04 (d, J = 8.2 Hz, 2H), 6.81 (s, 1H),

6.58 – 6.44 (m, 1H), 6.20 – 6.13 (m, 1H), 5.52 (s, 2H), 4.36 (bs, 4H), 3.75 (s, 3H), 3.43

(p, J = 7.5 Hz, 1H).

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Ethyl 1-((4-chlorobenzyl)carbamoyl)piperidine-4-carboxylate (31a). (4-

chlorobenzyl)isocyanate (0.30 mL, 2.26 mmol) was dissolved in DCM (10 mL) at RT,

then ethyl isonipecotate 26 (0.38 mL, 2.49 mmol) was added dropwise with stirring,

resulting in precipitation and discoloration. This slurry was allowed to stir at RT for 1 h,

at which time the precipitate was collected over a filter and dried via aspirator to afford

an off-white granular solid. No further purification was necessary. Yield: 690 mg, 2.13

mmol, 94%. 1H NMR (500 MHz, Chloroform-d) δ 7.30 – 7.25 (m, 2H), 7.25 – 7.20 (m,

2H), 4.84 (s, 1H), 4.37 (d, J = 5.6 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.87 (dt, J = 13.3,

3.7 Hz, 2H), 2.96 – 2.87 (m, 2H), 2.46 (ddd, J = 14.6, 10.7, 3.8 Hz, 1H), 1.91 (dd, J =

13.4, 3.1 Hz, 2H), 1.71 – 1.62 (m, 2H), 1.25 (t, J = 7.2 Hz, 3H).

Ethyl 1-((4-chlorophenethyl)carbamoyl)piperidine-4-carboxylate (31b). 1-

Chloro-4-(2-isocyanatoethyl)benzene 19 (0.45 mL, 2.94 mmol) was dissolved in DCM

(10 mL) at RT. Ethyl isonipecotate 26 was then added dropwise, which elicited

precipitation. The slurry was allowed to stir at RT for 1 h, at which time the precipitate

was collected and dried over a filter to afford ethyl 1-((4-chlorophenethyl)-

carbamoyl)piperidine-4-carboxylate as an off-white granular solid. This material was

taken into the next reaction in this crude form. Yield: 916 mg (92%). 1H NMR (500

MHz, Chloroform-d) δ 7.26 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.3 Hz, 2H), 4.52 (t, J = 5.3

Hz, 1H), 4.13 (q, J = 7.1 Hz, 2H), 3.79 (dt, J = 13.4, 3.8 Hz, 2H), 3.44 (q, J = 6.7 Hz,

2H), 2.90 – 2.80 (m, 2H), 2.78 (t, J = 6.9 Hz, 2H), 2.44 (tt, J = 10.8, 3.9 Hz, 1H), 1.92 –

1.83 (m, 2H), 1.62 (qd, J = 11.2, 4.1 Hz, 2H), 1.25 (t, J = 7.1 Hz, 3H).

119

Ethyl 1-(2-(4-chlorophenyl)acetyl)piperidine-4-carboxylate (33a). 4-

chlorophenylacetic acid (900 mg, 5.28 mmol) was dissolved in DCM (20 mL), followed

by the sequential addition of TEA (2.21 mL, 15.83 mmol), EDCHCl (1110 mg, 5.80

mmol), HOBT (889 mg, 5.80 mmol), and ethyl isonipecotate (0.894 mL, 5.80 mmol).

The reaction was stirred at RT for 28 h, after which time the DCM was evaporated off.

The residue was taken up in EtOAc and washed with 1M HCl (3X), H2O (5X), 10% aq.

Na2CO3 (3X), and brine (1X), then dried (MgSO4) and concentrated. The residue was

then purified through a plug of silica (30 g silica, 5% EtOAc:Hex). Yield: 1.31 g, 4.22

mmol, 80%. 1H NMR (500 MHz, Chloroform-d) δ 7.29 (d, J = 8.3 Hz, 2H), 7.17 (d, J =

8.2 Hz, 2H), 4.40 (dt, J = 12.5, 4.0 Hz, 1H), 4.13 (q, J = 7.1 Hz, 2H), 3.79 (dt, J = 13.5,

3.7 Hz, 1H), 3.69 (s, 2H), 3.12 – 3.05 (m, 1H), 2.89 – 2.82 (m, 1H), 2.52 – 2.45 (m, 1H),

1.96 – 1.87 (m, 2H), 1.83 – 1.77 (m, 1H), 1.61 – 1.57 (m, 1H), 1.49 (ddd, J = 24.1, 10.9,

3.8 Hz, 1H), 1.25 (t, J = 7.1 Hz, 3H).

Ethyl 1-(3-(4-chlorophenyl)propanoyl)piperidine-4-carboxylate (33b). 3-(4-

chlorophenyl)propionic acid (0.90 g, 4.87 mmol) was dissolved in DCM (20 mL),

followed by TEA (2.04 mL, 14.62 mmol), EDCHCl (1.03 g, 5.36 mmol), HOBT (0.82 g,

5.36 mmol), and ethyl isonipecotate (0.83 mL, 5.36 mmol). The solution was stirred at

RT for 28 h, after which time the DCM was removed in vacuo and the residue was

dissolved in 2:1 EtOAc:Et2O and washed with 1 M aq. HCl (3X), H2O (2X), 10% aq.

Na2SO3 (3X), and brine (1X), then dried (MgSO4) and concentrated. The residue was

then purified by silica flash chromatography (100 g silica, 5% to 50% EtOAc:Hex). 1H

NMR (500 MHz, Chloroform-d) δ 7.20 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.2 Hz, 2H), 4.41

120

– 4.34 (m, 1H), 4.10 (q, J = 7.1 Hz, 2H), 3.71 (d, J = 13.7 Hz, 1H), 3.04 – 2.96 (m, 1H),

2.89 (t, J = 7.8 Hz, 2H), 2.80 – 2.72 (m, 1H), 2.58 – 2.53 (m, 2H), 2.46 (tt, J = 10.7, 4.0

Hz, 1H), 1.90 – 1.80 (m, 2H), 1.54 (ddd, J = 24.1, 13.3, 3.5 Hz, 2H), 1.22 (t, J = 7.1 Hz,

3H).

1-Tert-Butyl 4-ethyl piperidine-1,4-dicarboxylate (34). Ethyl isonipecotate 26

(3.53 mL, 22.91 mmol), Boc2O (6.0 g, 27.50 mmol), and Na2CO3 (4.86 g, 45.80 mmol)

were added to a mixture of H2O (8 mL) and THF (20 mL) at RT (mild exotherms). This

was subsequently refluxed at 80 °C for 2 h, over which time the reaction proceeded from

a white suspension to a clear biphasic solution. The reaction was allowed to cool and was

extracted with EtOAc (2X) with no dilution of the aqueous phase. The organic extracts

were combined, dried (MgSO4), and concentrated in vacuo. The clear residue was then

purified by silica flash chromatography (150 g, 10% EtOAc:Hex) to give a thin, clear,

colorless liquid. Yield: 5.07 g, 19.70 mmol, 86%. 1H NMR (500 MHz, Chloroform-d) δ

4.12 (q, J = 7.1 Hz, 2H), 3.99 (s, 2H), 2.81 (t, J = 11.4 Hz, 2H), 2.41 (tt, J = 11.0, 3.6 Hz,

1H), 1.85 (d, J = 12.3 Hz, 2H), 1.60 (qd, J = 12.6, 12.1, 4.4 Hz, 2H), 1.43 (s, 9H), 1.23 (t,

J = 6.8 Hz, 3H).

Tert-Butyl 4-((2-(pyridin-4-yl)ethyl)carbamoyl)piperidine-1-carboxylate (35). The

boc-protected ethyl isonipecotate 34 (3.00 g, 11.66 mmol) was dissolved in EtOH (20

mL) and 10% aq. NaOH (10 mL) and stirred at RT for 4 h, after which time the EtOH

was stripped off in vacuo and the solution neutralized with conc. HCl at 0 ºC as quickly

as possible, and the resulting precipitate was collected over a filter and washed with cold

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H2O, then dried via aspirator, and finally high vacuum to provide the desired carboxylic

acid as a white powder. Yield: 2.43 g, 10.60 mmol, 91%.

The following was added to DCM: 1-(tert-butoxycarbonyl)piperidine-4-

carboxylic acid (600 mg, 2.62 mmol), DIPEA (1.37 mL, 7.85 mmol), EDC (552 mg, 2.88

mL), HOBT (441 mg, 2.88 mmol), and pyridylethylamine (0.34 mL, 2.88 mL). The

solution was stirred at RT for 17 h, at which time the DCM was stripped off and 10% aq.

sodium carbonate was added. Material was extracted out with EtOAc (3X). The organic

extractions were pooled, dried over magnesium sulfate, and concentrated in vacuo. The

residue was taken up in a small amount of EtOAc and diethyl ether was added. The

precipitate was collected over a filter and washed with diethyl ether to give the title

compound as an off-white solid. Yield: 634 mg, 1.9 mmol, 73%. 1H NMR (400 MHz,

Chloroform-d) δ 8.49 (d, J = 5.9 Hz, 2H), 7.09 (d, J = 5.9 Hz, 2H), 5.59 (t, J = 5.3 Hz,

1H), 4.22 – 3.95 (m, 2H), 3.52 (q, J = 6.8 Hz, 2H), 2.81 (t, J = 6.9 Hz, 2H), 2.68 (t, J =

12.5 Hz, 2H), 2.14 (tt, J = 11.6, 3.8 Hz, 1H), 1.77 – 1.65 (m, 2H), 1.56 (qd, J = 12.1, 4.3

Hz, 2H), 1.43 (s, 9H).

N-(2-(Pyridin-4-yl)ethyl)piperidine-4-carboxamide dihydrochloride (36). The

Boc-protected 35 (575 mg, 1.72 mmol) was suspended in Et2O at RT, and 4M HCl in

dioxane (6 mL, 24 mmol) was added. The mixture was stirred at RT for 30 min, at which

time the organic solution was decanted off and the solid material collected and dried in

vacuo. The title compound was thus obtained as a tan powder. Yield: 448 mg, 1.6 mmol,

97%. 1H NMR (400 MHz, DMSO-d6) δ 9.22 (bs, 1H), 8.91 (bs, 1H), 8.79 (d, J = 6.4 Hz,

2H), 8.18 (t, J = 5.6 Hz, 1H), 7.89 (d, J = 6.3 Hz, 2H), 3.40 (q, J = 6.2 Hz, 2H), 3.15 (d, J

122

= 12.6 Hz, 2H), 3.00 (t, J = 6.4 Hz, 2H), 2.83 – 2.69 (m, 2H), 2.39 – 2.26 (m, 1H), 1.80 –

1.59 (m, 4H). TOF ES+ MS: (M + H) 234.1, (M + Na) 256.1.

(R)-1-(4-Chlorobenzyl)pyrrolidine-2-carboxylic acid (R-38). L-proline (500 mg,

4.34 mmol) was dissolved in IPA (20 mL) in a dry flask, followed by the addition of

KOH pellets (730 mg, 13.03 mmol). Reaction was stirred at 40 °C while an IPA (5 mL)

solution of 4-chlorobenzyl chloride (770 mg, 4.78 mmol) was added via syringe pump

over 3 h. Reaction was allowed to stir an additional 6 h, after which time the reaction was

allowed to cool to RT. The solution was then acidified to pH 5 and DCM was added and

the reaction stirred for 16 hours at RT. After this time, the precipitate was filtered off and

washed with DCM (2X), and the combined organic filtrate extracts were concentrated to

provide a light yellow solid. Dried under high vacuum. Yield: 956 mg, 3.77 mmol, 87%.

1H NMR (400 MHz, Chloroform-d) δ 10.24 (s, 1H), 7.89 (d, J = 9.0 Hz, 1H), 7.35 (s,

4H), 6.62 (s, 2H), 4.75 (s, 4H), 1.58 (s, 2H). TOF ES– MS: (M – H) 238.1.

(S)-1-(4-Chlorobenzyl)pyrrolidine-2-carboxylic acid (S-38). D-proline (500 mg,


KOH pellets (730 mg, 13.03 mmol). Reaction was stirred at 40 °C while an IPA (5 mL)

solution of 4-chlorobenzyl chloride (770 mg, 4.78 mmol) was added via syringe pump

over 3 h. Reaction was allowed to stir an additional 6 h, after which time the reaction was

allowed to cool to RT. The solution was then acidified to pH 5 and DCM was added and

the reaction stirred for 16 hours at RT. After this time, the precipitate was filtered off and

washed with DCM (2X), and the combined organic filtrate extracts were concentrated to

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provide a light yellow solid. Taken directly into the subsequent step without further

characterization. Yield: 923 mg, 3.64 mmol, 84%. TOF ES– MS: (M – H) 238.1.

(R)-1-(4-Chlorobenzoyl)pyrrolidine-2-carboxylic acid (R-39). L-proline (800 mg,


KOH pellets (1.17 g, 20.85 mmol). Reaction was stirred at RT while an IPA solution of

4-chlorobenzoyl chloride (1.34 g, 7.64 mmol) was added via syringe pump over 1 h.

Reaction was allowed to stir an additional 1 h, after which time the reaction was cooled

to 0°C. The solution was then acidified to pH 5, DCM was added, and the reaction stirred

for 12 hours at RT. After this time, the precipitate was filtered off and washed with DCM

(2X), and the combined organic filtrate extracts were concentrated to provide an off-

white solid without further purification. Taken directly into the subsequent step without

further characterization. Yield: 1.58 g, 6.23 mmol, 90%. TOF ES– MS: (M – H) 252.0.

(S)-1-(4-Chlorobenzoyl)pyrrolidine-2-carboxylic acid (S-39). D-proline (800 mg,


KOH pellets (1.17 g, 20.85 mmol). Reaction was stirred at RT while an IPA solution of

4-chlorobenzoyl chloride (1.34 g, 7.64 mmol) was added via syringe pump over 1 h.

Reaction was allowed to stir an additional 1 h, after which time the reaction was cooled

to 0°C. The solution was then acidified to pH 5, DCM was added, and the reaction stirred

for 13 hours at RT. After this time, the precipitate was filtered off and washed with DCM

(2X), and the combined organic filtrate extracts were concentrated to provide an off-

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white solid without further purification. Taken directly into the subsequent step without

further characterization. Yield: 1.62 g, 6.39 mmol, 92%. TOF ES– MS: (M – H) 252.0.

(R)-Ethyl 1-(1-(4-chlorobenzoyl)pyrrolidine-2-carbonyl)piperidine-4-carboxylate

(R-40). (R)-1-(4-chlorobenzoyl)pyrrolidine-2-carboxylic acid (430 mg, 1.79 mmol) and

TEA (750 µL, 5.38 mmol) were dissolved in DCM (10 mL) followed by HATU (720 mg,

1.88 mmol), ethyl isonipecotate (290 µL, 1.88 mmol), and 3Å MS. The reaction was

allowed to stir at RT for 17 h, at which time the DCM was removed and the residue taken

up in a 1:1 solution of EtOAc:Et2O and washed with 0.5 M aq. HCl (2X), H2O (1X), 10%

aq. Na2CO3 (3X), and brine (1X). The solution was then dried (MgSO4) and

concentrated, and the residue was purified via silica flash chromatography (100 g silica,

50% to 100% EtOAc:Hex) to give the desired product as a clear oil. Yield: 420 mg, 1.11


(m, 2H), 5.04 (dt, J = 14.6, 7.4 Hz, 1H), 4.50 – 4.44 (m, 1H), 4.40 (m, 0.5H), 4.27 (dd, J

= 9.6, 3.9 Hz, 0.5H), 4.17 – 4.09 (m, 2H), 4.04 (d, J = 13.6 Hz, 0.5H), 3.94 (d, J = 13.7

Hz, 0.5H), 3.80 (tt, J = 11.5, 5.9 Hz, 0.5H), 3.67 (q, J = 7.8 Hz, 1H), 3.50 (ddd, J = 10.5,

7.2, 4.1 Hz, 1H), 3.41 – 3.30 (m, 0.5H), 3.17 (dt, J = 13.8, 6.9 Hz, 0.5H), 3.05 – 2.95 (m,

0.5H), 2.90 – 2.76 (m, 0.5H), 2.62 – 2.48 (m, 1H), 2.22 (ddt, J = 22.4, 15.3, 7.9 Hz, 1H),

2.06 – 1.81 (m, 7H), 1.64 (ddd, J = 27.7, 15.2, 8.6 Hz, 2H), 1.27 – 1.21 (m, 3H).

(S)-Ethyl 1-(1-(4-chlorobenzoyl)pyrrolidine-2-carbonyl)piperidine-4-carboxylate

(S-40). (S)-1-(4-chlorobenzoyl)pyrrolidine-2-carboxylic acid (490 mg, 2.05 mmol) and

TEA (860 µL, 6.15 mmol) were dissolved in DCM (10 mL) followed by HATU (820 mg,

125

2.15 mmol), ethyl isonipecotate (330 µL, 2.15 mmol), and 3Å MS. The reaction was

allowed to stir at RT for 16 h, at which time the DCM was removed and the residue taken

up in a 1:1 solution of EtOAc:Et2O and washed with 0.5 M aq. HCl (2X), H2O (1X), 10%

aq. Na2CO3 (3X), and brine (1X). The solution was then dried (MgSO4) and


50% to 100% EtOAc:Hex) to give the desired product as a clear oil. Yield: 340 mg, 0.91

mmol, 44%. 1H NMR (500 MHz, Chloroform-d) δ 7.52 (d, J = 8.1 Hz, 2H), 7.33 (m, 2H),

5.04 (dt, J = 14.6, 7.4 Hz, 1H), 4.49 – 4.40 (m, 1H), 4.27 (d, J = 9.6 Hz, 0.5H), 4.13 (dp,

J = 13.9, 7.4 Hz, 2H), 4.04 (d, J = 13.6 Hz, 0.5H), 3.94 (d, J = 13.7 Hz, 0.5H), 3.80 (tt, J

= 11.5, 5.9 Hz, 0.5H), 3.67 (q, J = 7.8 Hz, 1H), 3.50 (ddd, J = 10.5, 7.2, 4.1 Hz, 1H), 3.39

– 3.29 (m, 0.5H), 3.17 (dt, J = 13.8, 6.9 Hz, 0.5H), 3.05 – 2.94 (m, 0.5H), 2.88 – 2.80 (m,

0.5H), 2.61 – 2.48 (m, 1H), 2.22 (ddt, J = 22.4, 15.3, 7.9 Hz, 1H), 2.10 – 1.80 (m, 4H),

1.64 (m, 2H), 1.30 – 1.19 (m, 3H).

Methyl 2-(4-chlorophenoxy)benzoate (42). Methyl salicylate (0.40 mL, 3.86

mmol), pyridine (0.75 mL, 9.26 mmol), (4-chlorophenyl)boronic acid (531 mg, 3.40

mmol), and Cu(OAc)2 (617 mg, 3.40 mmol) were added to MeOH (10 mL) with 4Å MS

and allowed to stir at RT for 5 d. After this time, the reaction mixture was filtered over

celite and the filtrate was concentrated. The resulting residue was taken up in EtOAc and

washed with 5% aq. NaOH (3X) to remove residual copper, then H2O (3X), 1 M aq. HCl

(3X), and brine (1X), then the organic phase as dried (MgSO4) and concentrated, and the

resulting residue was purified via silica flash chromatography (1% to 10% EtOAc:Hex)

to provide the desired ether as colorless clear oil. Yield: 233 mg, 0.89 mmol, 23%. 1H

126

NMR (500 MHz, Chloroform-d) δ 7.90 (dd, J = 7.8, 1.6 Hz, 1H), 7.46 (ddd, J = 9.0, 7.9,

1.7 Hz, 1H), 7.29 – 7.24 (m, 2H), 7.23 – 7.17 (m, 1H), 6.96 (d, J = 8.3 Hz, 1H), 6.92 –

6.84 (m, 2H), 3.94 (s, 3H).

Methyl 2-((4-chlorophenyl)amino)benzoate (43a). Methyl salicylate (1.50 mL,

11.57 mmol), N,N-Bis(triflyl)aniline (4.76 g, 13.31 mmol), and DIPEA (6.06 mL, 34.70

mmol) were dissolved into anhydrous DMF (20 mL) in dry glassware under N2 at RT.

The solution was stirred for 18 h at RT, at which time the reaction was diluted with 2:1

EtOAc:Et2O and washed with H2O (5X) and brine (1X), then dried (MgSO4) and

concentrated. The residue was purified via silica flash chromatography (80 g silica, 5%

EtOAc:Hex) to give methyl 2-(((trifluoromethyl)sulfonyl)oxy)benzoate as a clear,

colorless oil. Yield: 2.76 g, 9.72 mmol, 84%. 1H NMR (500 MHz, Chloroform-d) δ 8.09

(d, J = 7.8 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.31 (d, J = 8.3 Hz,

1H), 3.97 (s, 3H).

A Schlenck flask was filled with toluene (60 mL, not anhydrous) and then

charged sequentially with the following reagents: methyl 2-

(((trifluoromethyl)sulfonyl)oxy)benzoate (900 mg, 3.17 mmol), 4-chloroaniline (485 mg,

3.80 mmol), Cs2CO3 (1444 mg, 4.43 mmol), Pd(OAc)2 (36 mg, 0.16 mmol), and DPPP

(104 mg, 0.25 mmol). The mixture was then thoroughly sparged with argon, and the flask

was fitted with a condenser and then purged and filled with an argon atmosphere. The

reaction was stirred at reflux under argon for 6 h, then allowed to cool to RT and stir for

12 h. At this time, the reaction was diluted with EtOAc and washed with 1M aq. HCl

(3X) and brine (1X), dried (MgSO4), and concentrated. The residue was purified via

127

silica flash chromatography (40 g silica, 1% to 5% EtOAc:Hex gradient) to yield the

desired compound as a clear, vaguely yellow oil. Yield: 710 mg, 2.71 mmol, 86%. 1H

NMR (500 MHz, Chloroform-d) δ 9.46 (s, 1H), 7.98 (d, J = 9.0 Hz, 1H), 7.37 – 7.26 (m,

3H), 7.24 – 7.16 (m, 3H), 6.77 (t, J = 7.6 Hz, 1H), 3.92 (d, J = 1.1 Hz, 3H).

Methyl 2-(phenylamino)benzoate (43b). Methyl salicylate (1.50 mL, 11.57

mmol), N,N-Bis(triflyl)aniline (4.76 g, 13.31 mmol), and DIPEA (6.06 mL, 34.70 mmol)

were dissolved into anhydrous DMF (20 mL) in dry glassware under N2 at RT. The

solution was stirred for 18 h at RT, at which time the reaction was diluted with 2:1

EtOAc:Et2O and washed with H2O (5X) and brine (1X), then dried (MgSO4) and

concentrated. The residue was purified via silica flash chromatography (80 g silica, 5%

EtOAc:Hex) to give methyl 2-(((trifluoromethyl)sulfonyl)oxy)benzoate as a clear,

colorless oil. Yield: 2.76 g, 9.72 mmol, 84%. 1H NMR (500 MHz, Chloroform-d) δ 8.09

(d, J = 7.8 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.31 (d, J = 8.3 Hz,

1H), 3.97 (s, 3H).

A Schlenck flask was filled with toluene (30 mL, not anhydrous) and then

charged sequentially with the following reagents: methyl 2-

(((trifluoromethyl)sulfonyl)oxy)benzoate (440 mg, 1.63 mmol), aniline (178 µL, 1.94

mmol), Cs2CO3 (743 mg, 2.28 mmol), Pd(OAc)2 (18 mg, 0.08 mmol), and DPPP (67 mg,

0.16 mmol). The mixture was then thoroughly sparged with argon, and the flask was

fitted with a condenser and then purged and filled with an argon atmosphere. The

reaction was stirred at reflux under argon for 6 h, then allowed to cool to RT and stir for

12 h. At this time, the reaction was diluted with EtOAc and washed with 1M aq. HCl

128

(3X) and brine (1X), dried (MgSO4), and concentrated. The residue was purified via

silica flash chromatography (40 g silica, 1% to 5% EtOAc:Hex gradient) to yield the

desired compound as a clear, somewhat yellow oil. Yield: 377 mg, 1.66 mmol, 57%. 1H

NMR (500 MHz, Chloroform-d) δ 9.47 (s, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.33 (d, J =

16.2, 7.3 Hz, 3H), 7.26 (d, J = 7.8 Hz, 3H), 7.10 (t, J = 7.3 Hz, 1H), 6.74 (t, J = 7.5 Hz,

1H), 3.91 (s, 3H).

Ethyl 1-(2-((4-chlorophenyl)amino)benzoyl)piperidine-4-carboxylate (44a).

Methyl 2-((4-chlorophenyl)amino)benzoate (650 mg, 2.48 mmol) was dissolved in 200

proof EtOH (15 mL) at RT and 10% aq. NaOH (6 mL) was added. The reaction was

allowed to stir at RT for 5 h, at which time the solvent was stripped off in vacuo and 1M

aq. HCl was added to the residue. The solution was then chilled in an ice bath and

acidified to pH 1, and the resulting white precipitate was collected over a filter, washed

with 1M aq. HCl, and dried via aspirator and high vacuum to yield a white powder. No

further purification necessary. Yield: 581 mg, 2.35 mmol, 94%. 1H NMR (500 MHz,

Chloroform-d) δ 9.27 (s, 1H), 8.06 (dd, J = 8.1, 1.5 Hz, 1H), 7.38 (ddd, J = 8.6, 7.1, 1.6

Hz, 1H), 7.35 – 7.31 (m, 2H), 7.23 – 7.19 (m, 2H), 7.17 (d, J = 8.5 Hz, 1H), 6.79 (t, J =

7.5 Hz, 1H).

2-((4-chlorophenyl)amino)benzoic acid (370 mg, 1.49 mmol) was then dissolved

in DCM (15 mL), followed by ethyl isonipecotate (253 µL, 1.64 mmol), TEA (625 µL,

4.48 mmol), EDCHCl (315 mg, 1.64 mmol), and HOBT (252 mg, 1.64 mmol). The

reaction was stirred at RT for 16 h, at which time the reaction was diluted with 2:1

EtOAc:Et2O and washed with 1 M aq. HCl (2X), 10% aq. Na2CO3 (3X), and brine (1X),

129

dried (MgSO4) and concentrated. The residue was purified via silica flash

chromatography (12 g silica, 5% to 20% EtOAc:Hex) to provide the product as a yellow

oil. Yield: 129 mg, 0.333 mmol, 22%. 1H NMR (500 MHz, Chloroform-d) δ 7.32 (d, J =

8.2 Hz, 1H), 7.25 – 7.15 (m, 4H), 7.02 (d, J = 8.7 Hz, 2H), 6.89 (t, J = 7.4 Hz, 1H), 4.30

(bs, 2H), 4.15 (q, J = 7.1 Hz, 2H), 3.09 (t, J = 11.1 Hz, 2H), 2.56 (tt, J = 10.6, 4.0 Hz,

1H), 1.95 (bs, 2H), 1.75 – 1.67 (m, 2H), 1.25 (t, J = 7.1 Hz, 3H).

Methyl 2-((4-chlorophenyl)(methyl)amino)benzoate (45). Methyl 2-((4-

chlorophenyl)amino)benzoate (150 mg, 0.57 mmol) was dissolved in anhydrous DMF (8

mL) under N2. The solution was then charged with granular K2CO3 (158 mg, 1.15 mmol).

MeI (40 µL, 0.63 mmol), dissolved in 1 mL anhydrous DMF, was added dropwise to the

solution at RT under N2, and the reaction was allowed to stir for 12 h at RT. After this

time, the reaction was diluted in 2:1 EtOAc:Et2O and washed with H2O (3X), 10% aq.

Na2S2O3 (1X), and brine (1X), then dried (MgSO4) and concentrated. The residue was

purified via silica flash chromatography (25 g silica, 1% to 10% EtOAc:Hex) to afford

the desired compound as a colorless oil taken crude into the next reaction. Yield: 139 mg,

0.50 mmol, 88%. TOF ES+ MS: (M + H) 276.1

Methyl 2-((4-chlorobenzyl)amino)benzoate (47a). Methyl 2-aminobenzoate (500

mg, 3.31 mmol) was dissolved in anhydrous DMF (15 mL) at RT under N2. Tert-BuOK

(408 mg, 3.64 mmol) was then added and the solution became yellow, and this was

allowed to stir at RT for 30 min, at which point 4-chlorobenzyl chloride (0.45 mL, 3.47

mmol) was added, and the solution lost color. The reaction was allowed to stir for 18 h

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under N2, at which time the reaction was diluted in EtOAc and washed with 10% aq.

NaHCO3 (1X), H2O (5X), and brine (1X), then dried (MgSO4) and concentrated. The

residue was purified by silica flash chromatography (40 g silica, 1% to 30% EtOAc:Hex)

to provide the desired compound as a colorless oil. Yield: 1.37 g, 2.88 mmol, 87%. 1H

NMR (500 MHz, Chloroform-d) δ 8.20 (t, J = 5.7 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.31

(q, J = 7.8, 7.1 Hz, 5H), 6.63 (t, J = 7.6 Hz, 1H), 6.58 (d, J = 8.5 Hz, 1H), 4.43 (d, J = 5.7

Hz, 2H), 3.88 (s, 3H). TOF ES+ MS: (M + H) 276.1.

Methyl 2-(benzylamino)benzoate (47b). To anhydrous DMF in dry glassware was

added methyl 2-aminobenzoate (280 mg, 1.85 mmol), benzyl bromide (330 mg, 1.94

mmol), and Cs2CO3 (1.50 g, 4.62 mmol). The reaction was stirred at 85 °C for 5 days,

after which time the reaction was cooled to RT and diluted with 1:1 EtOAc:Et2O and

washed with H2O (3X) and brine (1X), then dried (MgSO4) and concentrated. The

residue was purified via silica flash chromatography (5% EtOAc:Hex isochratic) to

separate product from unreacted starting material. Yield: 138 mg, 0.57 mmol, 31%. 1H

NMR (500 MHz, Chloroform-d) δ 8.18 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.40 – 7.25 (m,

6H), 6.65 (d, J = 8.5 Hz, 1H), 6.61 (t, J = 7.6 Hz, 1H), 4.46 (d, J = 5.6 Hz, 2H), 3.87 (s,

3H).

2-((4-Chlorophenethyl)amino)benzoic acid (49). 2-Bromobenzoate (200 mg, 1.00

mmol), 2-(4-chlorophenyl)ethanamine (290 mg, 1.86 mmol), K2CO3 (260 mg, 1.86

mmol), copper powder (12 mg, 0.19 mmol), and copper (I) oxide (13 mg, 0.09 mmol)

were added to ethylene glycol dimethyl ether (5 mL) in a dry pressure tube. The mixture

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was sparged and the headspace purged with argon, and the reaction was stirred at 130 °C

for 24 h. After cooling to RT, the reaction was diluted with EtOAc and H2O and filtered

through celite. The filtrate was acidified with conc. HCl and material was extracted with

EtOAc (3X). The combined extracts were dried (MgSO4) and concentrated. Purification

of the resulting residue was accomplished via silica flash chromatography (25 g silica,

10% EtOAc:Hex with 1% formic acid) to give a white powder. Yield: 120 mg, 0.44

mmol, 44%. 1

H NMR (500 MHz, Chloroform-d) δ 8.06 (d, J = 8.4 Hz, 1H), 8.00 (d, J =

8.0 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.32 (d, J = 8.2 Hz, 2H), 7.21 (d, J = 8.2 Hz, 2H),

6.71 (d, J = 8.5 Hz, 1H), 6.64 (t, J = 7.5 Hz, 1H), 3.47 (t, J = 7.1 Hz, 2H), 2.97 (t, J = 7.0

Hz, 2H).

2-(4-Chlorobenzoyl)benzoic acid (51). Aluminum chloride (9.0 g, 67.5 mmol)

was carefully added at RT to chlorobenzene (50 mL) in dry glassware with a water

separator for dense solvents, followed by phthalic anhydride (5.0 g, 33.8 mmol). The

reaction was then refluxed under N2 for 1.5 h, over which time it took on a deep red

color. Note: longer reaction times result in rapid accumulation of a second, internal

acylation. The reaction was then allowed to cool to RT over 1 h and was quenched by the

slow and careful addition of 1 M aq. HCl and allowed to stir another 1 h, over which time

color was lost and a white precipitate formed. The reaction was then taken up in EtOAc

and washed with 1M aq. HCl (1X), then the solvent was removed in vacuo. The residue

was then dissolved in 10% aq. NaOH, the resulting precipitate was filtered off, and the

filtrate was washed with EtOAc (2X) before being chilled in an ice bath and acidified to

pH 1 with conc. HCl. The resulting precipitate was collected over a filter to give the

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desired product as a white powder. No further purification necessary. Yield: 6.34 g, 24.31


(m, 3H), 7.58 (tt, J = 7.7, 1.2 Hz, 1H), 7.36 (dd, J = 12.2, 7.9 Hz, 3H). TOF ES– MS: (M

– H) 259.0.

2-(4-Chlorobenzyl)benzoic acid (52). 2-(4-chlorobenzoyl)benzoic acid 51 (200

mg, 0.77 mmol) was added to 28% aq. ammonia (10 mL), followed by zinc (600 mg,

9.21 mmol) and CuSO45H2O (80 mg, 0.31 mmol). This mixture was stirred at reflux for

48 h, with 2 mL recharges of 28% aq. ammonia every 6 h, barring 12 h at night. After

this time, the reaction was allowed to cool to RT and the solution was decanted off the

solid precipitate and concentrated in vacuo. The residue was taken up in H2O and

acidified to pH < 1 by conc. HCl, then extracted 1X with EtOAc. The extract was dried

(MgSO4) and concentrated, and the residue was crystallized from EtOAc to give tan

crystals that were used immediately in the subsequent reaction. Yield: 62 mg, 0.25 mmol,

33%. TOF ES– MS: (M – H) 245.0.

Methyl 6-chloro-9H-carbazole-1-carboxylate (55). 9H-carbazole-1-carboxylic

acid 54 (200 mg, 0.95 mmol) – prepared in a manner similar to that reported120

– was

stirred in refluxing sat. methanolic HCl for 1 h. The reaction was then allowed to cool

and was concentrated. The residue was dissolved in EtOAc and washed with sat. aq.

Na2CO3 (1X), then dried (MgSO4), and concentrated. The residue was then purified by

silica flash chromatography (10 g silica, 10% EtOAc/Hex) to give a white powder. Yield:

196 mg, 0.87 mmol, 92%. 1H NMR (500 MHz, Chloroform-d) δ 9.94 (s, 1H), 8.26 (d, J =

133

7.6 Hz, 1H), 8.14 – 8.04 (m, 2H), 7.57 – 7.46 (m, 2H), 7.34 – 7.28 (m, 1H), 7.26 (t, J =

7.7 Hz, 1H), 4.03 (s, 3H).

Methyl 9H-carbazole-1-carboxylate (90 mg, 0.40 mmol) was stirred in DCM (15

mL) at 0 °C under N2 while SO2Cl2 was added dropwise. The reaction was allowed to stir

at 0 °C for 4 h. At this time, the reaction was diluted with DCM and washed with 10%

aq. Na2CO3 (2X) and brine (1X), then dried (Na2SO4) and concentrated. The residue was

purified via silica flash chromatography (10 g silica, 10 to 30% EtOAc/Hex) to give a

white powder. Yield: 75 mg, 0.29 mmol, 72%. 1H NMR (500 MHz, Chloroform-d) δ 9.93

(s, 1H), 8.21 (d, J = 7.7 Hz, 1H), 8.10 (d, J = 7.6 Hz, 1H), 8.04 (s, 1H), 7.44 – 7.41 (m,

2H), 7.27 (t, J = 7.7 Hz, 1H), 4.03 (s, 3H). 1H NMR (500 MHz, Benzene-d6) δ 9.70 (s,

1H), 8.03 (d, J = 7.6 Hz, 1H), 7.84 (d, J = 1.9 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.17 (dd,

J = 8.6, 2.0 Hz, 1H), 6.92 (t, J = 7.7 Hz, 1H), 6.46 (d, J = 8.6 Hz, 1H), 3.48 (s, 3H).

Ethyl 1-(2-oxo-1,2-dihydroquinolin-4-yl)piperidine-4-carboxylate (57). 4-

chloroquinolin-2(1H)-one was prepared according to literature precedent.124, 125

4-

chloroquinolin-2(1H)-one (500 mg, 2.78 mmol), ethyl isonipecotate 26 (0.52 mL), NaI

(420 mg, 2.78 mmol), and Na2CO3 (885 mg, 8.35 mmol) were added to DMF (10 mL) at

RT under N2 and stirred at 80 °C for 48 h. The reaction was then cooled to RT, at which

time it was diluted in 1:1 EtOAc:Et2O and washed with 1 M HCl (3X), H2O (3X), 10%

aq. Na2CO3 (3X), and brine (1X). The organic phase was dried (MgSO4) and

concentrated. The residue was purified by silica flash chromatography (45 g silica, 20 to

80% EtOAc/Hex) to give a white microcrystalline solid. Yield: 567 mg, 1.89 mmol, 68%.

1H NMR (500 MHz, Chloroform-d) δ 12.43 (s, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.48 – 7.40

134

(m, 2H), 7.17 (t, J = 7.2 Hz, 1H), 6.13 (s, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.54 (d, J = 12.4

Hz, 2H), 2.82 (t, J = 10.8 Hz, 2H), 2.54 (ddd, J = 15.0, 10.8, 4.1 Hz, 1H), 2.13 – 2.07 (m,

2H), 2.06 – 1.98 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H).

Ethyl 1-(1-(4-chlorophenyl)-2-oxo-1,2-dihydroquinolin-4-yl)piperidine-4-

carboxylate (58). Ethyl 1-(2-oxo-1,2-dihydroquinolin-4-yl)piperidine-4-carboxylate 57

(490 mg, 1.64 mmol) was dissolved in DCM (10 mL) with 4 Å MS, followed by the

addition of 4-chlorophenylboronic acid (510 mg, 3.27 mmol), pyridine (0.26 mL, 3.27

mmol), TEA (0.46 mL, 3.27 mmol), and Cu(OAc)2 (590 mg, 3.27 mmol). The reaction

was stirred at RT for 4 d, over which time the reaction became increasingly green with

precipitate. After this time, the reaction was diluted with more DCM and washed with

10% aq. NaOH (3X), 10% aq. NaHCO3 (1X), and brine (1X), then dried (MgSO4) and

concentrated. The resulting residue was purified by silica flash chromatography (25 g, 5

to 50% EtOAc/Hex) to give a white solid. Yield: 480 mg, 1.18 mmol, 72%. 1H NMR

(500 MHz, Chloroform-d) δ 7.79 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 8.3 Hz, 2H), 7.32 (t, J =

7.8 Hz, 1H), 7.23 (d, J = 8.3 Hz, 2H), 7.19 (t, J = 7.6 Hz, 1H), 6.66 (d, J = 8.5 Hz, 1H),

6.21 (s, 1H), 4.21 (q, J = 7.1 Hz, 2H), 3.55 (d, J = 12.2 Hz, 2H), 2.84 (t, J = 11.5 Hz, 2H),

2.57 (ddd, J = 15.1, 10.9, 4.1 Hz, 1H), 2.16 – 2.09 (m, 2H), 2.09 – 1.96 (m, 2H), 1.31 (t, J

= 7.1 Hz, 3H).

Ethyl 1-(1-(4-chlorobenzyl)-2-oxo-1,2-dihydroquinolin-4-yl)piperidine-4-

carboxylate (59). Ethyl 1-(2-oxo-1,2-dihydroquinolin-4-yl)piperidine-4-carboxylate 58

(95 mg, 0.32 mmol) was dissolved in anhydrous DMF (5 mL) under nitrogen and cooled

135

to 0 °C. KOtBu (43 mg, 0.38 mmol) was added, which elicited a slight color change. 4-

chlorobenzyl chloride 4a (49 µL, 0.38 mmol) was then added and the reaction was

allowed to stir at RT for 15 h. At this time, the reaction was diluted with 2:1 EtOAc:Et2O

and washed with 1M aq. HCl (3X), H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X),

then dried (MgSO4) and concentrated. Residue was purified via silica flash

chromatography (10 g silica, 1% to 30% EtOAc:Hex). Yield: 117 mg, 0.28 mmol, 87%.

1H NMR (500 MHz, Chloroform-d) δ 7.79 (d, J = 7.9 Hz, 1H), 7.40 (t, J = 7.7 Hz, 1H),

7.28 – 7.23 (m, 2H), 7.17 (dd, J = 13.3, 8.2 Hz, 4H), 6.24 (s, 1H), 5.48 (s, 2H), 4.20 (q, J

= 7.3 Hz, 2H), 3.52 (d, J = 12.4 Hz, 2H), 2.82 (t, J = 11.8 Hz, 2H), 2.55 (tt, J = 11.0, 8.8,

4.0 Hz, 1H), 2.06 (ddd, J = 31.9, 19.5, 6.8 Hz, 4H), 1.30 (t, J = 7.4 Hz, 3H).

Ethyl 1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)piperidine-4-carboxylate (70).

The indole carboxylic acid 4a (982 mg, 3.44 mmol) was added to DCM (15 mL),

followed by ethyl isonipecotate 26 (0.80 mL, 5.16 mmol), DIPEA (1.80 mL, 10.31

mmol), EDCHCl (800 mg, 4.12 mmol), and HOBT (630 mg, 4.12 mmol). The reaction

was allowed to stir at RT for 24 h, after which time it was diluted with 1:1 EtOAc:Et2O

and washed with 1M HCl (5X), H2O (5X), 10% aq. Na2CO3 (5X) and brine (1X), then

dried (MgSO4) and concentrated. The residue can then be crystallized from a two-phase

organic solution of EtOAc and Hex to give crystals. Yield: 1.22 g, 2.88 mmol, 83%. 1H


– 7.27 (m, 1H), 7.25 – 7.20 (m, 2H), 7.19 – 7.14 (m, 1H), 7.08 – 6.97 (m, 2H), 6.68 –

6.61 (m, 1H), 5.49 (d, J = 2.5 Hz, 2H), 4.49 (bs, 2H), 4.17 (qd, J = 7.1, 2.5 Hz, 2H), 3.08

136

– 2.95 (m, 2H), 2.51 (tq, J = 9.4, 3.6 Hz, 1H), 1.97 – 1.55 (m, 4H), 1.28 (td, J = 7.2, 2.5

Hz, 3H).

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)piperidine-4-carboxylic acid (71).

The ethyl ester 70 (1.22 g, 2.88 mmol) was dissolved in EtOH (20 mL) and 5M aq.

NaOH (20 mL) and stirred at 50 °C for 48 h. Most of the solvent was then removed in

vacuo and the remaining aqueous solution was washed with Et2O (1X), then acidified to

pH<1 at 0 °C with conc. HCl. The material was extracted with 1:1 EtOAc:Et2O (3X), and

the combined extracts were dried (MgSO4) and concentrated. The resulting residue was

dissolved in EtOAc and triturated with Hex, collected over a filter, and dried to give the

desired carboxylic acid as white, fluffy microcrystals. Yield: 1.03 g, 2.59 mmol, 90%. 1H


– 7.23 (m, 1H), 7.19 – 7.11 (m, 3H), 6.99 (d, J = 8.0 Hz, 2H), 6.59 (s, 1H), 5.42 (s, 2H),

4.38 (bs, 1H), 4.08 (bs, 1H), 2.87 (bs, 2H), 2.38 (bs, J = 11.0 Hz, 1H), 1.90 – 1.37 (m,

4H).

2,3-Dihydro-1H-indene-2-carboxamide (74). 2-indancarboxylic acid 73 (1.4 g,

8.63 mmol) was dissolved in anhydrous DMF (20 mL), and the following was added

sequentially: DIPEA (3.02 mL, 17.26 mmol), HATU (3.94 g, 10.36 mmol), and HMDS

(2.17 mL, 10.36 mmol) after 30 min of stirring at RT. After stirring for 20 h at RT, the

solution was taken up in EtOAc and washed with 0.5 M HCl (3X), H2O (3X), and 10%

aq. Na2CO3 (3X). The organic phase was collected and dried over MgSO4s, and

concentrated in vacuo. The solid residue was recrystallized from EtOH to afford the title

137

compound as sharp, colorless crystals. Yield: 861 mg, 5.06 mmol, 59%. 1H NMR (400

MHz, DMSO-d6) δ 7.40 (bs, 1H), 7.22 – 7.15 (m, 2H), 7.14 – 7.09 (m, 2H), 6.86 (bs,

1H), 3.19 – 3.10 (m, 1H), 3.06 – 3.02 (m, 4H).

(2,3-Dihydro-1H-inden-2-yl)methanamine (75). 2,3-dihydro-1H-indene-2-

carboxamide 74 (55 mg, 0.341 mmol) was added to anhydrous THF (8 mL), and cooled

in an ice bath under nitrogen. LAH (0.34 mL, 0.341 mmol, 1M THF soln) was added

under nitrogen, and the reaction was allowed to warm to rt and stir for 7 h. At this time,

the reaction was quenched by the Fieser method and the precipitate removed over a filter.

The filtrate was collected and concentrated in vacuo, and then taken up in a small amount

of THF. 4M HCl/dioxane was added, the solvent removed in vacuo, and the residue

sonicated in Et2O to afford the easily handled hydrochloride salt. Yield: 1H NMR (400

MHz, Chloroform-d) δ 7.22 – 7.16 (m, 2H), 7.16 – 7.10 (m, 2H), 3.08 (dd, J = 15.6, 8.0

Hz, 2H), 2.80 (d, J = 7.1 Hz, 2H), 2.66 (dd, J = 15.6, 7.0 Hz, 2H), 2.54 (p, J = 7.4 Hz,

1H). TOF ES+ MS: (M + H) 148.1, (M + Na) 170.1.

1-(2,3-Dihydro-1H-inden-2-yl)ethanone (77). Acetylacetate (97 µL, 0.95 mmol)

was dissolved in anhydrous DMF under N2, followed by the addition of 60% oil-

suspended NaH (95 mg, 2.37 mmol). This was allowed to stir for 30 min, at which time

1,2-bis(bromoethyl)benzene 76 (250 mg, 0.95 mmol) was added. The reaction was

allowed to stir for 1 h at RT, then 8 h at 100 °C, then allowed to cool to RT. The reaction

was quenched with sat. aq. NH4Cl, diluted with EtOAc, and washed with 10% aq.

NaHCO3 (3X) and brine (1X), then dried (MgSO4) and concentrated. The residue was

138

then purified via silica flash chromatography (50 g silica, 1% to 5% EtOAc:Hex) to give

a very greasy solid invisible to electrospray MS. Yield: 1H NMR (400 MHz, Chloroform-

d) δ 7.21 – 7.17 (m, 2H), 7.17 – 7.12 (m, 2H), 3.47 – 3.37 (m, 1H), 3.20 – 3.11 (m, 4H),

2.22 (d, J = 2.6 Hz, 3H).

1-(2,3-Dihydro-1H-inden-2-yl)ethanamine (78). 1-(2,3-dihydro-1H-inden-2-

yl)ethanone 77 (44 mg, 0.275 mmol) was dissolved in methanol (2 mL), and 7 M

methanolic ammonia was added (1 mL, 7 mmol). This was allowed to stir at RT for 4 h,

at which time NaBH4 (31 mg, 0.824 mmol) and a catalytic drop of glacial AcOH was

added. Stirring continued for 14 h at RT, after which time the solvent was removed in

vacuo. The residue was taken up in EtOAc and washed with a small amount of 10% aq.

Na2CO3, dried with MgSO4, and concentrated. The residue was then taken on in

subsequent reactions without further purification or characterization. TOF ES+ MS: (M +

H) 162.2.

Ethyl 7-methyl-1H-indole-2-carboxylate (81). To a 0 °C flask charged with

crushed ice (~10 g) was added o-toluidine 79 (6.00 mL, 55.4 mmol), followed by conc.

HCl (10 mL) which elicited the precipitation of the hydrochloride salt. Dissolved in H2O

(30 mL), NaNO2 (3.82 g, 55.4 mmol) was added dropwise slowly to the flask, and more

ice was added as needed. The reaction was allowed to stir at 0 °C for 30 min, after which

~15 g of crushed ice and a 0 °C slurry of SnCl2 (32 g, 166.0 mmol) in conc. HCl (10 mL)

was added. The reaction was then allowed to sit in a fridge (4 °C) for 11 h, after which

time the precipitate was filtered off and collected, and washed with cold brine and

139

hexanes. The resulting precipitate was collected over a filter and washed with cold brine

and 1:1 EtOAc:Hex. Unlike other reports, no attempt was made to basify, wash, and re-

convert the hydrazine to the hydrazine hydrochloride salt (due to observed stability

issues). The white solid was dried thoroughly under high vacuum. Yield: 4.92 g, 31.02

mmol, 56%.

Ethyl pyruvate 80 (1.17 mL, 10.51 mmol), o-tolylhydrazine hydrochloride (1.5 g,

9.46 mmol), ZnCl2 (7.16 g, 52.55 mmol) and TsOH (9.05 g, 52.55 mmol) were dissolved

in anhydrous EtOH (15 mL) at RT in dry glassware. The reaction was refluxed for 8 h,

after which the reaction was allowed to cool to RT and the resulting precipitate was

filtered off and washed with EtOH. The filtrate was concentrated to give a residue that

was dissolved in 1:1 EtOAc:Et2O and washed with 10% aq. Na2CO3 (2X), dried

(MgSO4), and concentrated. The residue was then purified by silica flash chromatography

(100 g silica, 20% EtOAc:Hex) to give the desired indole as a foul-smelling oil. Yield:

1.37 g, 6.72 mmol, 71%. 1H NMR (400 MHz, Chloroform-d) δ 8.88 (bs, 1H), 7.53 (d, J =

8.0 Hz, 1H), 7.27 – 7.21 (m, 1H), 7.14 – 7.02 (m, 2H), 4.42 (qd, J = 7.1, 1.3 Hz, 2H),

2.52 (s, 3H), 1.42 (td, J = 7.1, 1.3 Hz, 3H).

Ethyl 1-(4-chlorobenzyl)-7-methyl-1H-indole-2-carboxylate (82). Ethyl 7-methyl-

1H-indole-2-carboxylate 81 (500 mg, 2.46 mmol) was dissolved in anhydrous DMF (20

mL) and granular K2CO3 (425 mg, 3.08 mmol) and 4-chlorobenzyl chloride (0.39 mL,

3.08 mmol) were added. The mixture was stirred for 3 d at 60 °C, after which time the

incomplete reaction was terminated by dilution with EtOAc and subsequent washing with

brine (3X). The organic phase was dried over MgSO4, concentrated, and purified vial

140

silica flash chromatography (80 g silica, 40% EtOAc:Hex) to provide the title compound

as white crystals. Yield: 390 mg, 1.19 mmol, 46%. 1H NMR (400 MHz, Chloroform-d) δ

7.59 – 7.53 (m, 1H), 7.43 – 7.40 (m, 1H), 7.20 (d, J = 8.4 Hz, 2H), 7.06 – 6.97 (m, 2H),

6.78 (d, J = 8.2 Hz, 2H), 6.08 (s, 2H), 4.28 (q, J = 7.1 Hz, 2H), 2.54 (s, 3H), 1.33 (t, J =

7.1 Hz, 3H).

1-(4-Chlorobenzyl)-7-methyl-1H-indole-2-carboxylic acid (83). Ethyl 1-(4-

chlorobenzyl)-7-methyl-1H-indole-2-carboxylate 82 (212 mg, 0.647 mmol) was

dissolved in EtOH (10 mL) and 10% aq. KOH (10 mL) was added. The reaction stirred

for 4 h at 70 °C, after which time as much solvent was stripped off as possible in vacuo.

The mostly aqueous mixture was cooled in an ice bath and acidified with conc. HCl to

obtain a precipitate that was collected over a filter and washed with 1 M HCl and cold

H2O. No further purification necessary. Yield: 182 mg, 0.607 mmol, 94%. 1H NMR (400

MHz, DMSO-d6) δ 12.94 (s, 1H), 7.36 – 7.24 (m, 4H), 7.01 – 6.95 (m, 2H), 6.76 (d, J =

8.1 Hz, 2H), 6.08 (s, 2H), 2.44 (s, 3H).

Ethyl 3-methyl-1H-indole-2-carboxylate (86). Phenylhydrazine was prepared in a

manner similar to that reported.138

Ethyl 2-oxobutanoate 85 (500 mg, 4.90 mmol),

phenylhydrazine (0.44 mL, 4.41 mmol), and TsOH (2.3 g, 12.24 mmol) were dissolved in

anhydrous EtOH (15 mL) at RT in dry glassware. The reaction was refluxed for 12 h,

over which time it took on a dark hue. After the reaction time, the reaction was allowed

to cool to RT and the resulting precipitate was filtered off and washed with EtOH. The

combined filtrate was concentrated to give a residue that was dissolved in 1:1

141

EtOAc:Et2O and washed with 10% aq. Na2CO3 (2X), dried (MgSO4), and concentrated.

The residue was then purified by silica flash chromatography (45 g silica, 20%

EtOAc:Hex) to give the desired indole as a foul-smelling oil. Yield: 646 mg, 3.18 mmol,

65%. 1H NMR (500 MHz, Chloroform-d) δ 8.83 (s, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.39

(d, J = 8.3 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.16 (t, J = 7.4 Hz, 1H), 4.45 (qd, J = 7.1,

1.1 Hz, 2H), 2.64 (d, J = 1.1 Hz, 3H), 1.46 (td, J = 7.2, 1.1 Hz, 3H).

Ethyl 1-(4-chlorobenzyl)-3-methyl-1H-indole-2-carboxylate (87). Anhydrous

DMF (12 mL) in dry glassware was charged with ethyl 3-methyl-1H-indole-2-

carboxylate 86 (646 mg, 3.18 mmol), 4-chlorobenzyl chloride (0.50 mL, 3.97 mmol), and

granular K2CO3, and the mixture was stirred at 60 °C for 14 h, allowed to cool for 1 h,

then diluted in 1:1 EtOAc:Et2O and washed with 10% aq. Na2CO3 (3X), dried (MgSO4),

and concentrated. The residue was purified by silica flash chromatography ( 40 g

silica, 20% EtOAc:Hex) to give the desired compound as an oil. Yield: 750 mg, 2.29

mmol, 72%. 1H NMR (400 MHz, Chloroform-d) δ 7.71 (dd, J = 8.1, 1.1 Hz, 1H), 7.33 –

7.23 (m, 2H), 7.22 – 7.12 (m, 3H), 6.99 – 6.90 (m, 2H), 5.73 (s, 2H), 4.34 (qd, J = 7.2,

1.1 Hz, 2H), 2.62 (d, J = 1.1 Hz, 3H), 1.35 (td, J = 7.1, 1.1 Hz, 3H).

5-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)octahydro-1H-pyrrolo[3,4-

c]pyridin-1-one (91). The following were added sequentially to DMF (10 mL): 1-(4-

chlorobenzyl)-1H-indole-2-carboxylic acid 4a (250 mg, 0.875 mmol), DIPEA (0.46 mL,

2.64 mmol), EDCHCl (202 mg, 1.050 mmol), and HOBT (162 mg, 1.050 mmol). This

solution was allowed to stir at RT for 1 h, at which time crude octahydro-1H-pyrrolo[3,4-

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c]pyridin-1-one 90 (123 mg, 0.875 mmol) was added, having been prepared through the

known route.140

Stirring continued for 12 h at RT, at which time the solution was

partitioned between water and 1:1 solution of EtOAc:Et2O. The organic extract was then

washed with water (1X) and saturated aq. Na2CO3 (1X), dried with MgSO4, and

concentrated in vacuo. Purification was accomplished via silica flash chromatography

(100 g silica, 100% EtOAc.) Resulting residue was crystallized from the in vacuo

removal of DCM to give the title compound as a slightly pink solid. Yield: 200 mg, 0.49


(m, 4H), 7.19 (t, J = 7.4 Hz, 1H), 7.14 (d, J = 8.1 Hz, 2H), 6.93 (s, 1H), 6.31 (bs, 1H),

5.51 (s, 2H), 4.57 (d, J = 13.2 Hz, 1H), 4.18 (bs, 1H), 3.56 – 3.46 (m, 1H), 3.39 (dd, J =

13.7, 5.1 Hz, 1H), 3.05 (d, J = 8.6 Hz, 1H), 2.63 (s, 1H), 2.50 (s, 1H), 1.96 – 1.73 (m,

2H), 1.54 (bs, 1H).

Tert-Butyl 4-(3-(pyridin-4-yl)propanamido)piperidine-1-carboxylate (94). t-Butyl

4-aminopiperidine-1-carboxylate (500 mg, 2.50 mmol), 3-(pyridin-4-yl)propanoic acid

(377 mg, 2.50 mmol), TEA (1.04 mL, 7.49 mmol), EDCHCl (574 mg, 3.00 mmol), and

HOBT (459 mg, 3.00 mmol) were all dissolved in DCM (30 mL) and stirred at RT for 30

h, after which time the DCM was removed in vacuo and the residue was taken up in

EtOAc and washed with H2O (1X), 10% Na2CO3 (3X), and brine (1X), then dried

(MgSO4) and concentrated. The resulting residue was purified via silica flash

chromatography (40 g silica, 1% to 10% methanolic ammonia:EtOAc) to provide a white

solid. Yield: 591 mg, 1.773 mmol, 71%.

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Ethyl 2-(1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)piperidin-4-yl)acetate (96).

Indole carboxylic acid 4a (520 mg, 1.82 mmol), ethyl 2-(piperidin-4-yl)acetate 95 (345

mg, 1.65 mmol), TEA (1.15 mL, 8.27 mmol), EDCHCl (380 mg, 1.98 mmol), and

HOBT (304 mg, 1.98 mmol) were all dissolved in DCM (50 mL) and stirred at RT for 24

h, after which time the DCM was removed in vacuo and the residue was taken up in

EtOAc and washed with 10% aq. citric acid (3X), H2O (2X), 10% Na2CO3 (3X), and

brine (1X), then dried (MgSO4) and concentrated. The resulting residue was purified via

silica flash chromatography (40 g silica, 10% to 50% EtOAc:Hex) to provide a white

powder. Yield: 460 mg, 1.05 mmol, 63%. 1H NMR (500 MHz, Chloroform-d) δ 7.67 –

7.62 (m, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.29 – 7.25 (m, 1H), 7.24 – 7.20 (m, 2H), 7.18 –

7.13 (m, 1H), 7.06 – 7.01 (m, 2H), 6.62 (s, 1H), 5.48 (s, 2H), 4.63 (bs, 1H), 4.13 (q, J =

7.2 Hz, 3H), 2.88 (bs, 1H), 2.74 (bs, 1H), 2.18 (d, J = 7.0 Hz, 2H), 1.98 (ttt, J = 11.0, 7.1,

3.7 Hz, 1H), 1.75 (bs, 1H), 1.57 (bs, 1H), 1.26 (t, J = 7.1 Hz, 3H), 1.11 (bs, 1H), 0.65 (bs,

1H).

4-(3-(Triphenylphosphoranyl)propyl)pyridine bromide (98). 4-(3-

hydroxypropyl)pyridine 97 (1.42 g, 10.35 mmol) and PPh3 (4.07 g, 15.53 mmol) were

dissolved in conc. HBr (8 mL) in a pressure vessel. The reaction was sealed and the

suspension as stirred at 135 °C for 2 h, over which time a homogenous solution was

achieved. After this time, the reaction was allowed to cool to RT, eliciting precipitation.

The reaction was taken up in toluene and transferred to a flask equipped with a Dean-

Stark apparatus and water condenser, and H2O was removed with heating of 150 °C for 3

h, at which time more conc. HBr (8 mL) was added. H2O was again removed at 150 °C

144

for 6 h. Once sufficient water had been removed, a precipitate began to appear and the

reaction was allowed to cool to RT, at which point it was diluted with H2O (50 mL) and

washed with Et2O (5X). The pH was raised to pH 7 by the addition of 10% aq. NaOH,

and the resulting solution was washed with Et2O to remove PPh3 which caused

significant precipitation of product. The aqueous slurry was then washed with Et2O (3X)

and cooled in a fridge for 2 h, after which time the precipitate was collected over a filter

and washed with cold H2O and Et2O. The highly hygroscopic crystalline material was

dried under high vacuum for 1 d and stored in a desiccator. No further purification

necessary. Off-white crystalline solid. Yield: 1.601 g, 3.46 mmol, 34%. 1H NMR (500

MHz, Chloroform-d) δ 8.41 (d, J = 5.9 Hz, 2H), 7.82 – 7.74 (m, 9H), 7.70 – 7.64 (m,

6H), 7.17 (d, J = 5.9 Hz, 2H), 3.98 – 3.90 (m, 2H), 3.11 (t, J = 7.4 Hz, 2H), 1.93 (dq, J =

15.7, 7.8 Hz, 2H). TOF ES+ MS: (M+) 382.2.

(Z)-tert-Butyl 4-(4-(pyridin-4-yl)but-1-en-1-yl)piperidine-1-carboxylate (99a).

Due to the poor ethereal solubility of the phosphonium bromide, the reaction was

conducted at RT. 4-(3-(bromotriphenylphosphoranyl)propyl)pyridine 98 (330 mg, 0.714

mmol) was added to anhydrous THF in a flame-dried flask under N2 at RT. To the

suspension, n-BuLi (2.5 M in hexane) was added dropwise at RT until a light yellow

color persisted, at which point a full equivalent was added (285 µL, 0.714 mmol) to give

a red solution. The reaction then stirred for 10 min at RT under N2 to allow for ylide

dissolution. Predissolved in anhydrous THF (10 mL), t-butyl 4-formylpiperidine-1-

carboxylate 8 (138 mg, 0.649 mmol) was added dropwise at RT to the reaction, and the

reaction was allowed to stir at RT under N2 for 14 h, at which time the reaction was

145

quenched with sat. NH4Cl and extracted with a 1:1 solution of EtOAc:Et2O . This extract

was then washed with 10% aq. Na2CO3 (1X) and brine (1X), dried (MgSO4) and

concentrated. The residue was purified via silica flash chromatography (12 g silica,

isocratic 60% EtOAc:Hex) to give a clear, colorless oil, >50:1 Z:E. Yield: 117 mg, 0.370


5.1 Hz, 2H), 5.38 (t, J = 6.1 Hz, 1H), 5.36 (d, J = 5.9 Hz, 1H), 4.04 (s, 2H), 2.67 (q, J =

10.3, 7.7 Hz, 4H), 2.31 (q, J = 7.1 Hz, 2H), 2.08 – 1.97 (m, 1H), 1.64 – 1.55 (m, 2H),

1.45 (s, 9H), 1.19 (tt, J = 13.2, 5.9 Hz, 2H). TOF ES+ MS: (M + H, no Boc) 217.2.

(E)-tert-Butyl 4-(4-(pyridin-4-yl)but-1-en-1-yl)piperidine-1-carboxylate (99b).

LiBr (340 mg, 3.92 mmol) was dried by flame-heating a 2-neck flask under high vacuum

until the molten salt ceased bubbling. The flask was then purged with N2 and a stir bar

was added (so the bar would not char during the flame-dry) and the flask was allowed to

cool to RT. The flask was then charged with anhydrous THF (40 mL) and 4-(3-

(triphenylphosphoranyl)propyl)pyridine bromide 98 (823 mg, 1.78 mmol) under N2.

Without cooling – due to the poor ethereal solubility of the phosphonium bromide – 2 M

PhLi in (Bu)2O was added at RT until a slight yellow color persisted, at which point a full

1.1 equivalent of PhLi (0.98 mL, 1.96 mmol) was added, and the suspension gave way to

mostly homogenous red solution after 20 min of RT stirring. This solution was then

chilled to -78 °C, and t-butyl 4-formylpiperidine-1-carboxylate 8 (380 mg, 1.78 mmol) –

pre-dissolved in anhydrous THF (10 mL) – was added dropwise to the solution, causing a

loss of color. The light yellow solution was allowed to stir at -78 °C for 30 min, at which

time the second equivalent of PhLi (0.98 mL, 1.96 mmol) was added dropwise, resulting

146

in the solution becoming a very dark cherry-red color. The cooling bath was then

removed, and the reaction was allowed to stir for 30 min as it approached RT. The

reaction was then re-chilled to -78 °C and t-BuOH (0.204 mL, 2.14 mmol) – predissolved

in anhydrous THF (5 mL) – was added dropwise, eliciting precipitation and

decolorization. The reaction was allowed to warm to RT and stir for 36 h, after which

time the reaction was quenched with a small amount of sat. aq. NH4Cl and diluted with a

2:1 EtOAc:Et2O solution and washed with 10% aq. Na2CO3 (1X) and brine (1X), dried

(MgSO4), and concentrated in vacuo. Purification accomplished by silica flash

chromatography (40 g silica, 50% to 80% EtOAc:Hex) to provide the desired olefin as a

clear colorless oil, ~5:1 E:Z. Yield: 231 mg, 0.730 mmol, 41%. 1H NMR (400 MHz,

Chloroform-d) δ 8.43 (d, J = 4.4 Hz, 2H), 7.06 (d, J = 4.5 Hz, 2H), 5.25 (t, J = 8.9 Hz,

1H), 5.16 (t, J = 10.0 Hz, 1H), 3.98 (s, 2H), 2.67 – 2.55 (m, 4H), 2.34 (q, J = 7.3 Hz, 2H),

2.27 – 2.12 (m, 1H), 1.40 (s, 9H), 1.30 (s, 2H), 1.13 (d, J = 11.8 Hz, 2H). TOF ES+ MS:

(M + H, no Boc) 217.2, (M + Na, no Boc) 239.2.

Tert-Butyl 4-((2-(pyridin-4-yl)ethyl)carbamoyl)piperazine-1-carboxylate (101). t-

Butyl piperazine-1-carboxylate (400 mg, 2.15 mmol) was dissolved in DCM (15 mL) at

RT under N2 and cooled to 0 °C. 15% wt. phosgene (360 µL, 0.50 mmol) in toluene was

added to the solution dropwise, followed by the dropwise addition of TEA (0.90 mL, 6.44

mmol), and then the dropwise addition of 4-(2-aminoethyl)pyridine (565 µL, 4.72 mmol).

The reaction was allowed to stir for 1 h at 0 °C, at which point it was diluted with EtOAc

and washed with 10% aq. NaOH (1X), H2O (3X), and brine (1X), dried (MgSO4), and

concentrated. The residue was then purified via silica flash chromatography (40 g silica,

147

1% to 20% methanolic ammonia:EtOAc) to give a white solid after concentration. Yield:

582 mg, 1.74 mmol, 81%. 1H NMR (500 MHz, Chloroform-d) δ 8.44 (d, J = 5.6 Hz, 2H),

7.09 (d, J = 5.4 Hz, 2H), 4.92 (t, J = 5.8 Hz, 1H), 3.47 (q, J = 6.7 Hz, 2H), 3.39 – 3.34

(m, 5H), 3.30 – 3.27 (m, 3H), 2.81 (t, J = 7.0 Hz, 2H), 1.43 (d, J = 1.6 Hz, 12H).

1-Tert-Butyl 4-ethyl 4-(phenylselanyl)piperidine-1,4-dicarboxylate (103). DIPA

(2.49 mL, 17.49 mmol) was dissolved into anhydrous THF (10 mL) in dry glassware

under N2, then chilled to -78 °C. Then, 2.5 M n-BuLi (5.60 mL, 13.99 mmol) was added

dropwise, and the reaction was allowed to stir for 10 min. 1-Tert-butyl 4-ethyl piperidine-

1,4-dicarboxylate 102 (3.00 g, 11.66 mmol) – pre-dissolved in THF (10 mL) – was added

dropwise to the reaction and the reaction was allowed to stir at -78 °C for 15 min. PhSeCl

(2.68 g, 13.99 mmol) – pre-dissolved in anhydrous THF (10 mL) – was added dropwise

to the reaction under the same conditions. The cooling bath was removed to allow for

warming to RT, and the reaction was stirred for 3 h. After this time, the reaction was

quenched by the addition of sat. aq. NH4Cl and extracted with 1:1 EtOAc:Et2O. The

extract was subsequently washed with H2O (2X) and brine (1X), dried (MgSO4) and

concentrated. The residue was purified by silica flash chromatography (80 g, 5%

EtOAc:Hex) to give a clear, slightly yellow oil. Yield: 2.80 g, 6.78 mmol, 58%. 1H NMR

(500 MHz, Chloroform-d) δ 7.56 (d, J = 7.5 Hz, 2H), 7.39 (t, J = 7.4 Hz, 1H), 7.30 (t, J =

7.7 Hz, 2H), 4.10 (q, J = 7.1 Hz, 2H), 3.74 (s, 2H), 3.11 – 3.03 (m, 2H), 2.13 – 2.06 (m,

2H), 1.84 (s, 2H), 1.44 (s, 9H), 1.18 (t, J = 7.1 Hz, 3H).

148

Ethyl 4-(phenylselanyl)piperidine-4-carboxylate hydrochloride (104). 1-tert-butyl

4-ethyl 4-(phenylselanyl)piperidine-1,4-dicarboxylate (1.70 g, 4.12 mmol) was dissolved

in Et2O (100 mL) at RT and 4 M HCl in 1,4-dioxane (15.46 mL, 61.80 mmol) was added

dropwise. The reaction was stirred at RT for 4 d until complete by TLC. The solvent was

removed in vacuo and Et2O was added and stripped off five times (5X) to remove excess

HCl and solidify the salt. Further removal of solvent and HCl via rotovap and, lastly, high

vacuum furnished the hydrochloride salt. Yield, 1.44, g, 4.12 mmol, 100%. TOF ES+

MS: (M + H) 314.1, (M + Na) 336.0.

Ethyl 1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)-4-(phenylselanyl)piperidine-

4-carboxylate (105). Ethyl 4-(phenylselanyl)piperidine-4-carboxylate hydrochloride (200

mg, 0.574 mmol) was dissolved in DCM (10 mL) and TEA (0.48 mL, 3.44 mmol),

followed by 1-(4-chlorobenzyl)-1H-indole-2-carboxylic acid (164 mg, 0.574 mmol),

EDCHCl (121 mg, 0.631 mmol), and HOBT (97 mg, 0.631 mmol), and the reaction was

allowed to stir at RT for 15 h. After this time, the reaction was diluted with a 1:1 solution

of EtOAc:Et2O and washed with 1M HCl (3X), H2O (3X), 10% Na2CO3 (3X), and brine

(1X), then dried (MgSO4) and concentrated in vacuo. The resulting residue was purified

via silica flash chromatography (12 g silica, 5% to 20% EtOAc:Hex) to yield an oil that

may triturated to provide a fine white powder. Yield: 208 mg, 0.359 mmol, 63%. 1H


– 7.36 (m, 2H), 7.30 (dt, J = 15.2, 7.8 Hz, 3H), 7.23 (d, J = 8.3 Hz, 2H), 7.16 (t, J = 7.5

Hz, 1H), 7.04 (d, J = 8.2 Hz, 2H), 6.61 (s, 1H), 5.48 (s, 2H), 4.12 (q, J = 7.1 Hz, 3H),

149

3.84 (s, 1H), 3.33 – 3.20 (m, 2H), 2.12 (s, 1H), 1.81 (s, 2H), 1.45 (s, 1H), 1.19 (t, J = 7.1

Hz, 3H).

Ethyl 1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)-1,2,3,6-tetrahydropyridine-4-

carboxylate (106). Ethyl 1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)-4-

(phenylselanyl)piperidine-4-carboxylate (160 mg, 0.276 mmol) was dissolved in DCM

(10 mL) at RT then cooled to -78 °C. Suspended in DCM (10 mL), ~60% mCPBA (80

mg, 0.276 mmol) was added to the solution dropwise and the reaction was allowed to stir

for 30 min. Still at -78 °C, TEA (115 µL, 0.828 mmol) was added dropwise and the

reaction was allowed to stir for another 30 min. At this time, the reaction was warmed to

RT, then the reaction was then diluted with 2:1 EtOAc:Et2O and washed with 10% aq.

Na2CO3 (3X), H2O (1X), 1 M HCl (2X), H2O (1X) again, and sat. aq. Na2CO3 (1X). The

organic phase was dried with MgSO4 and concentrated. The resulting solid residue was

taken up in Hex and the yellow supernatant was decanted off to remove selenoxides.

Further purification was accomplished by silica flash chromatography (12 g silica, 5% to

20% EtOAc:Hex) to provide the desired unsaturated ester as a white solid. Yield: 96 mg,

0.227 mmol, 82%. 1H NMR (500 MHz, Chloroform-d) δ 7.66 (d, J = 7.9 Hz, 1H), 7.37

(d, J = 8.3 Hz, 1H), 7.29 (t, J = 7.7 Hz, 1H), 7.22 – 7.11 (m, 3H), 7.00 (d, J = 7.5 Hz,

2H), 6.68 (s, 1H), 5.48 (s, 2H), 4.23 (q, J = 7.1 Hz, 4H), 3.68 (s, 2H), 2.32 (s, 2H), 1.31

(t, J = 7.1 Hz, 3H).

1-Tert-Butyl 4-ethyl 4-methylpiperidine-1,4-dicarboxylate (107). DIPA (0.62 mL,

4.37 mmol) was dissolved into anhydrous THF (10 mL) in dry glassware under N2, then

150

chilled to -78 °C. Then, 2.5 M n-BuLi (1.40 mL, 3.50 mmol) was added dropwise, and

the reaction was allowed to stir for 15 min. Boc-ethyl isonipecotate 102 (750 mg, 2.91

mmol) – pre-dissolved in THF (5 mL) – was added dropwise to the reaction and the

reaction was allowed to stir at -78 °C for 15 min. MeI (220 µL, 3.50 mmol) – pre-

dissolved in anhydrous THF (3 mL) – was added dropwise to the reaction under the same

conditions. The cooling bath was removed to allow for warming to RT, and the reaction

was stirred for 8 h. After this time, the reaction was quenched by the addition of sat. aq.

NH4Cl and diluted with 1:1 EtOAc:Et2O, then washed with H2O (3X) and brine (1X),

dried (MgSO4) and concentrated. The residue was purified by silica flash

chromatography (40 g, 5 to 20% EtOAc:Hex) to give a clear colorless oil. 1H NMR (500

MHz, Chloroform-d) δ 4.14 (q, J = 7.1 Hz, 2H), 3.74 (s, 2H), 2.96 (s, 2H), 2.04 (d, J =

13.3 Hz, 2H), 1.42 (s, 9H), 1.32 (t, J = 12.5 Hz, 2H), 1.24 (t, J = 7.5 Hz, 3H), 1.17 (s,

3H).

Ethyl 4-methylpiperidine-1,4-dicarboxylate hydrochloride (108). The Boc-

protected amino ester 107 (500 mg, 1.84 mmol) was dissolved into 4M HCl dioxane (4

mL) and stirred for 15 min. After this time, the dioxane was removed in vacuo and the

material was sonicated in Et2O to give a tan solid. The Et2O was decanted off and the

precipitate was dried via aspirator, then high vacuum, to afford the desired amine

hydrochloride as a tan, hygroscopic solid. No further purification necessary. Yield: 350

mg, 1.68 mmol, 100%. TOF ES+ MS: (M + H) 172.1, (M + Na) 194.1.

151

Ethyl 1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)-4-methylpiperidine-4-

carboxylate (109). The amine hydrochloride 108 (240 mg, 1.16 mmol), indole carboxylic

acid 4a (300 mg, 1.05 mmol), TEA (585 µL, 4.20 mmol), EDCHCl (242 mg, 1.26

mmol), and HOBT (193 mg, 1.26 mmol) were dissolved in DCM (30 mL) and stirred at

RT for 22 h. After this time, the reaction as diluted with EtOAc and washed with 1M HCl

(3X), H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X). The organic extract was dried

(MgSO4) and concentrated, and the residue purified via silica flash chromatography (40 g

silica, 30% to 90% EtOAc:Hex) to give a white solid. Yield: 281 mg, 0.64 mmol, 61%.

1H NMR (500 MHz, Chloroform-d) δ 7.65 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H),

7.31 – 7.26 (m, 1H), 7.21 (d, J = 7.3 Hz, 2H), 7.16 (t, J = 7.5 Hz, 1H), 7.03 (d, J = 7.8

Hz, 2H), 6.61 (s, 1H), 5.48 (s, 2H), 4.29 (bs, 1H), 4.18 (q, J = 7.4, 7.0 Hz, 2H), 3.86 (bs,

1H), 3.10 (bs, 1H), 2.98 (bs, 1H), 2.12 (bs, 1H), 1.85 (bs, 1H), 1.31 – 1.20 (m, 4H), 1.14

(s, 3H), 0.77 (s, 2H).

(1R,5S,6r)-Ethyl 3-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)-3-

azabicyclo[3.1.0]hexane -6-carboxylate (113). The amine exo-112 (170 mg, 0.89 mmol)

– prepared in a manner similar to that reported150, 151

– indole carboxylic acid 4a (304 mg,

1.06 mmol), TEA (495 µL, 3.55 mmol), EDCHCl (204 mg, 1.06 mmol), and HOBT (163

mg, 1.06 mmol) were dissolved in DCM (10 mL) and stirred at RT for 18 h. After this

time, the reaction as diluted with EtOAc:Et2O (1:1) and washed with 1M HCl (1X), H2O

(2X), 10% aq. Na2CO3 (3X), and brine (1X). The organic extract was dried (MgSO4) and

concentrated, and the residue purified via silica flash chromatography (25 g silica, 5% to

20% EtOAc:Hex) to give a white solid. Yield: 244 mg, 0.58 mmol, 78%. 1H NMR (500

152

MHz, Chloroform-d) δ 7.66 (d, J = 7.9 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.32 – 7.28 (m,

1H), 7.26 – 7.22 (m, 2H), 7.17 (dd, J = 8.2, 6.7 Hz, 1H), 6.99 (d, J = 8.1 Hz, 2H), 6.74 (s,

1H), 5.56 (d, J = 49.7 Hz, 2H), 4.22 (bd, J = 12.4 Hz, 1H), 4.15 (q, J = 7.2 Hz, 2H), 3.77

(bd, J = 13.0 Hz, 2H), 3.52 (s, 1H), 2.18 – 2.04 (m, 2H), 1.29 (td, J = 7.1, 1.2 Hz, 3H),

1.06 (t, J = 3.1 Hz, 1H).

(1R,3s,5S)-Tert-Butyl 3-((2-(pyridin-4-yl)ethyl)carbamoyl)-8-

azabicyclo[3.2.1]octane-8-carboxylate (115). The Boc-protected amino ester 114 (175

mg, 0.65 mmol) was dissolved in anhydrous THF (10 mL) with KOTMS (83 mg, 0.65

mmol) and stirred under dry conditions under N2 for 18 h, at which time a precipitate was

observed and the solvent was removed in vacuo to give the potassium salt as a white

powder. No further purification necessary. Yield: 190 mg, 0.65 mmol, 100%.

The potassium salt (191 mg, 0.65 mmol), amine 27 (93 µL, 0.78 mmol),

EDCHCl (150 mg, 0.78 mmol), HOBT (119 mg, 0.78 mmol), and TEA (362 µL, 2.60

mmol) were dissolved in DCM (10 mL) and stirred at RT for 24 h, at which time the

DCM was removed in vacuo and the residue was taken up in EtOAc and washed with

H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X). The organic extract was dried (MgSO4)

and concentrated, and the residue purified via silica flash chromatography (25 g silica,

1% to 20% methanolic ammonia:EtOAc) to give a tan powder. Yield: 41 mg, 0.083

mmol, 57%. Yield: 146 mg, 0.41 mmol, 63%. 1H NMR (500 MHz, Chloroform-d) δ 8.49

(d, J = 5.8 Hz, 2H), 7.10 (d, J = 5.7 Hz, 2H), 5.66 (t, J = 5.9 Hz, 1H), 4.23 (d, J = 40.3

Hz, 2H), 3.54 – 3.48 (m, 2H), 2.81 (t, J = 7.0 Hz, 2H), 2.58 (tt, J = 11.8, 5.3 Hz, 1H),

1.97 – 1.84 (m, 5H), 1.61 – 1.56 (m, 3H), 1.46 (d, J = 1.1 Hz, 9H).

153

N-(2-(Pyridin-4-yl)ethyl)-2-tosyl-2-azaspiro[3.3]heptane-6-carboxamide (123).

The tosyl amide carboxylic acid 122 (2.0 g, 6.77 mmol) – prepared in a manner similar to

that reported152, 153

– amine 27 (0.97 mL, 8.13 mmol), HATU (309 g, 8.13 mmol), and

TEA (2.83 mL, 20.31 mmol) were dissolved in DMF (10 mL) and stirred at RT for 18 h,

at which time the reaction was diluted with EtOAc and washed with H2O (5X), 10% aq.

Na2CO3 (3X), and brine (1X). The organic extract was dried (MgSO4) and concentrated,

and the residue purified via silica flash chromatography (80 g silica, 1% to 20%

methanolic ammonia:EtOAc) to give an extremely thick, clear, yellow-tinged oil. Yield:

1.95 g, 4.88 mmol, 72%. 1H NMR (500 MHz, Chloroform-d) δ 8.31 (d, J = 5.4 Hz, 2H),

7.59 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 5.4 Hz, 2H), 6.30 (t, J =

5.8 Hz, 1H), 3.61 (d, J = 5.5 Hz, 4H), 3.37 (q, J = 6.8 Hz, 2H), 2.69 (t, J = 7.1 Hz, 2H),

2.62 (p, J = 7.8 Hz, 1H), 2.37 (s, 3H), 2.12 (dd, J = 11.1, 8.7 Hz, 2H), 2.05 – 1.98 (m,

2H). TOF ES+ MS: (M + H) 400.1, (M + Na) 422.1.

Methyl 1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)azetidine-3-carboxylate

(125). The following reagents were dissolved in DCM (10 mL) at RT: indole carboxylic

acid 4a (150 mg, 0.53 mmol), methyl 4-azetidinecarboxylate hydrochloride (88 mg, 0.58

mmol), TEA (366 µL, 2.26 mmol), EDCHCl (121 mg, 0.63 mmol), and HOBT (96 mg,

0.63 mmol). The reaction was allowed to stir for 26 h at RT, after which time the solvent

was remove in vacuo and the residue was dissolved in EtOAc and washed with 1M HCl

(3X), H2O (2X), 10% aq. Na2CO3 (3X), and brine (1X), then dried (MgSO4) and

concentrated. The residue was purified by silica flash chromatography (25 g silica, 5 to

154

40% EtOAc:Hex). Yield: 117 mg, 0.30 mmol, 58%. 1H NMR (500 MHz, Chloroform-d)

δ 7.67 (d, J = 8.0 Hz, 1H), 7.34 – 7.27 (m, 2H), 7.22 – 7.14 (m, 3H), 7.04 – 6.99 (m, 2H),

6.83 (d, J = 1.2 Hz, 1H), 5.73 (s, 2H), 4.53 (bd, J = 36.9 Hz, 2H), 4.32 (bd, J = 26.0 Hz,

2H), 3.78 (d, J = 1.4 Hz, 3H), 3.47 (p, J = 7.1 Hz, 1H).

Ethyl 1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)azepane-4-carboxylate (132).

The carboxylic acid 4a (757 mg, 2.65 mmol), azapane ester hydrochloride 131 (550 mg,

2.65 mmol) – prepared in a manner similar to that reported160

– EDCHCl (558 mg, 2.91

mmol), HOBT (446 mg, 2.91 mmol), and TEA (1.48 mL, 10.59 mmol) were dissolved in

DCM (20 mL) and stirred at RT for 18 h, at which time the DCM was removed in vacuo

and the residue was taken up in EtOAc and washed with 1M HCl (2X), H2O (3X), 10%

aq. Na2CO3 (3X), and brine (1X). The organic extract was dried (MgSO4) and


20% methanolic ammonia:EtOAc) to give a colorless oil. Yield: 756 mg, 1.72 mmol,

65%. 1H NMR (500 MHz, Chloroform-d) δ 8.09 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.2 Hz,

1H), 7.74 (d, J = 8.1 Hz, 0.5H), 7.66 (d, J = 8.0 Hz, 0.5H), 7.59 (ddd, J = 8.2, 7.0, 1.1 Hz,

1H), 7.47 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.30 – 7.25 (m, 0.5H),

7.23 – 7.16 (m, 1.5H), 7.04 (d, J = 8.0 Hz, 1H), 6.99 (d, J = 8.1 Hz, 0.5H), 6.68 (d, J =

11.9 Hz, 0.5H), 6.37 (d, J = 1.2 Hz, 2H), 5.59 – 5.42 (m, 1H), 4.16 (ddt, J = 19.2, 14.0,

7.2 Hz, 2H), 3.98 – 3.60 (m, 2.5H), 3.51 (d, J = 12.2 Hz, 1H), 3.42 – 3.25 (m, 1H), 2.79

(s, 0.5H), 2.53 (d, J = 47.4 Hz, 0.5H), 2.41 (s, 0.5H), 2.20 – 2.04 (m, 1H), 1.94 (bs,

1.5H), 1.77 (bd, J = 12.1 Hz, 1H), 1.63 (m, 0.5H), 1.51 (bs, 0.5H), 1.38 (m, J = 10.0 Hz,

0.5H), 1.33 – 1.01 (m, 3H).

155

Methyl 1-(4-chlorobenzyl)-4-fluoro-1H-pyrrole-2-carboxylate (138). Methyl 4-

fluoro-1H-pyrrole-2-carboxylate 137 (130 mg, 0.91 mmol) was dissolved in anhydrous

DMF at room temperature, followed by the addition of potassium carbonate (151 mg,

1.09 mmol) and 2a (0.14 mL, 1.09 mmol). The reaction was then stirred for 30 h at 60

°C, after which time it was allowed to cool to room temperature. The reaction was diluted

with a 1:1 solution of ethyl acetate/diethyl ether, washed with water (3×) and brine (1×),

dried with magnesium sulfate, and concentrated in vacuo. The resulting brown residue

was purified via flash chromatography (60 g silica, 10% ethyl acetate/hexanes) to afford

methyl 1-(4-chlorobenzyl)-4-fluoro-1H-pyrrole-2-carboxylate as a light-yellow oil.

Yield: 203 mg (83%). 1H NMR (400 MHz, CDCl3) δ 7.27 (d, J = 8.3 Hz, 2H), 7.03 (d, J

= 8.3 Hz, 2H), 6.69−6.59 (m, 2H), 5.44 (s, 2H), 3.75 (s, 3H).

1-(4-Chlorobenzyl)-4-fluoro-1H-pyrrole-2-carboxylic acid (139). Methyl 1-(4-

chlorobenzyl)-4-fluoro-1H-pyrrole-2-carboxylate 138 (150 mg, 0.56 mmol) was

dissolved in ethanol (10 mL) at room temperature. Then 10% aqueous sodium hydroxide

(2 mL) was added, and the reaction was stirred for 18 h at room temperature. At this time,

solvent was stripped off in vacuo until material began to precipitate. Additional water

was added (2 mL), and the solution was cooled in an ice bath, then acidified with

concentrated HCl. The resulting precipitate was collected over a filter and washed with

cold 1 M HCl, then dried under high vacuum to afford the desired carboxylic acid as a

white powder. Yield: 111 mg (78%). 1H NMR (400 MHz, CDCl3) δ 7.29 (d, J = 8.3 Hz,

2H), 7.04 (d, J = 8.3 Hz, 2H), 6.79 (d, J = 2.0 Hz, 1H), 6.71−6.65 (m, 1H), 5.43 (s, 2H).

156

Ethyl 1-(4-chlorobenzyl)-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (144). Ethyl 1-

(4-chlorobenzyl)-1H-pyrrolo[2,3-c]pyridine-2-carboxylate 143 (300 mg, 0.953 mmol)

was dissolved into ethanol (8 mL) at RT, followed by the addition of 10% aqueous NaOH

(8 mL), which initially caused precipitation, but homogeneity was achieved over time.

The reaction was allowed to stir at RT for 12 h, at which time the solvent was removed in

vacuo. The residue was taken up in a small amount of water (5 mL), the pH was adjusted

to ∼4, and material was extracted with ethyl acetate (8×). The extracts were combined

and the solvent removed again in vacuo to provide the title compound as a fine white

powder. Yield: 226 mg, 83%. 1H NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H), 8.40 (d, J =

6.3 Hz, 1H), 8.26 (d, J = 6.3 Hz, 1H), 7.61 (s, 1H), 7.35 (d, J = 8.5 Hz, 2H), 7.10 (d, J =

8.5 Hz, 2H), 6.06 (s, 2H).

(4-(Cyclohexylmethyl)piperazin-1-yl)(1-(4-fluorobenzyl)-1H-indol-2-

yl)methanone (CCG-203941). The following reagents were dissolved in anhydrous DMF

(1.5 mL) at RT: 1-(4-fluorobenzyl)-1H-indole-2-carboxylic acid 4b (50 mg, 0.186

mmol), 1-(cyclohexylmethyl)piperazine (34 mg, 0.186 mmol), DIPEA (97 µL, 0.557

mmol), EDCHCl (46 mg, 0.241 mmol), and HOBT (37 mg, 0.241 mmol). The reaction

was allowed to stir for 24 h at RT, after which time it was diluted with 1:1 EtOAc:Et2O

and washed with 10% aq. Na2CO3 (2X), then dried (MgSO4) and concentrated. The

residue was purified by silica flash chromatography (20 g silica, 60% EtOAc:Hex).


Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.32 – 7.29 (m, 1H), 7.20 – 7.16 (m, 1H), 7.11 (dd, J =

157

8.6, 5.4 Hz, 2H), 6.95 (t, J = 8.7 Hz, 2H), 6.64 (s, 1H), 5.51 (s, 2H), 3.65 (bd, J = 74.0

Hz, 4H), 2.36 (s, 2H), 2.11 – 1.95 (m, 4H), 1.79 – 1.67 (m, 5H), 1.31 – 1.16 (m, 4H), 0.89

– 0.80 (m, 2H). HPLC tR = 6.65 min, >95% purity.

(4-Benzylpiperazin-1-yl)(1-(4-fluorobenzyl)-1H-indol-2-yl)methanone (CCG-

203942). The following was added sequentially to anhydrous DMF: 1-(4-fluorobenzyl)-

1H-indole-2-carboxylic acid (50 mg, 0.186 mmol), DIPEA (65 µL, 0.371 mmol),

EDCHCl (46 mg, 0.241 mmol), HOBT (37 mg, 0.241 mmol), and 1-benzylpiperazine

(33 mg, 0.186 mmol). The solution was allowed to stir at room temperature for 22 h. At

this time, a 1:1 solution of EtOAc:Et2O (5 mL) was added, and this was washed with

aqueous 10% Na2CO3 (3X). The extract was then dried with anhydrous MgSO4, filtered,

and the filtrate was concentrated in vacuo. The crude residue was purified by column

chromatography (20 g silica, 80% EtOAC/Hexanes) to provide the title compound. Yield:

57 mg, 0.13 mmol, 72 %. 1H NMR (500 MHz, Chloroform-d) δ 7.68 (d, J = 7.9 Hz, 1H),

7.43 (d, J = 8.3 Hz, 1H), 7.39 – 7.31 (m, 6H), 7.20 (t, J = 7.5 Hz, 1H), 7.12 (dd, J = 7.8,

5.6 Hz, 2H), 6.98 (t, J = 8.5 Hz, 2H), 6.66 (s, 1H), 5.53 (s, 2H), 3.68 (bd, J = 67.9 Hz,

4H), 3.49 (s, 2H), 2.21 (bd, 4H). HPLC tR = 5.91 min, > 95%.

(4-Benzoylpiperidin-1-yl)(1-(4-fluorobenzyl)-1H-indol-2-yl)methanone (CCG-

203943). The following reagents were dissolved in anhydrous DMF (2 mL) at RT: 1-(4-

fluorobenzyl)-1H-indole-2-carboxylic acid (50 mg, 0.186 mmol), phenyl(piperidine-4-

yl)methanone hydrochloride (42 mg, 0.186 mmol), DIPEA (72 µL, 0.557 mmol),

EDCHCl (46 mg, 0.241 mmol), and HOBT (37 mg, 0.241 mmol). The reaction was

158

allowed to stir for 24 h at RT, after which time it was diluted with 1:1 EtOAc:Et2O and

washed with 10% aq. Na2CO3 (3X), then dried (MgSO4) and concentrated. The residue

was purified by silica flash chromatography (20 g silica, 80% EtOAc:Hex). Yield: 68 mg,

0.154 mmol, 83%. 1H NMR (500 MHz, Chloroform-d) δ 7.96 (dt, J = 8.4, 1.2 Hz, 2H),

7.72 – 7.65 (m, 1H), 7.64 – 7.58 (m, 1H), 7.54 – 7.49 (m, 2H), 7.41 (d, J = 8.3 Hz, 1H),

7.30 (ddd, J = 8.5, 7.1, 1.1 Hz, 1H), 7.19 (td, J = 7.4, 1.0 Hz, 1H), 7.16 – 7.11 (m, 2H),

7.02 – 6.93 (m, 2H), 6.69 (d, J = 0.9 Hz, 1H), 5.52 (s, 2H), 4.63 (bs, 1H), 4.20 (bs, 1H),

3.51 (tt, J = 10.7, 3.7 Hz, 1H), 3.06 (s, 2H), 2.05 – 1.48 (m, 4H). HPLC tR = 8.32 min,

>95%.

(4-(Benzyloxy)piperidin-1-yl)(1-(4-fluorobenzyl)-1H-indol-2-yl)methanone

(CCG-203944). The following reagents were dissolved in anhydrous DMF (3 mL) at RT:

1-(4-fluorobenzyl)-1H-indole-2-carboxylic acid (50 mg, 0.186 mmol), 4-

(benzyloxy)piperidine hydrochloride (42 mg, 0.186 mmol), DIPEA (72 µL, 0.557 mmol),


allowed to stir for 24 h at RT, after which time it was diluted with 1:1 EtOAc:Et2O and

washed with 10% aq. Na2CO3 (3X), then dried (MgSO4) and concentrated. The residue

was purified by silica flash chromatography (20 g silica, 80% EtOAc:Hex). Yield: 29 mg,

0.066 mmol, 35%. 1H NMR (500 MHz, Chloroform-d) δ 7.70 (d, J = 7.9 Hz, 1H), 7.45 –

7.37 (m, 5H), 7.36 – 7.28 (m, 2H), 7.21 (ddd, J = 7.9, 7.0, 0.9 Hz, 1H), 7.16 – 7.11 (m,

2H), 7.00 – 6.94 (m, 2H), 6.68 (d, J = 0.8 Hz, 1H), 5.53 (s, 2H), 4.58 (s, 2H), 3.92 (bd, J

= 66.0 Hz, 2H), 3.66 (tt, J = 7.2, 3.5 Hz, 1H), 3.47 (bd, J = 48.5 Hz, 2H), 1.97 – 1.42 (m,

4H). HPLC tR = 8.86 min, >95% purity.

159

(1-(4-Fluorobenzyl)-1H-indol-2-yl)(4-(piperidine-1-carbonyl)piperidin-1-

yl)methanone (CCG-203945). The following was added sequentially to anhydrous DMF

(2 mL): 1-(4-fluorobenzyl)-1H-indole-2-carboxylic acid (50 mg, 0.186 mmol), DIPEA

(0.065 ml, 0.371 mmol), EDCHCl (46.3 mg, 0.241 mmol), HOBT (37.0 mg, 0.241

mmol), and piperidin-1-yl(piperidin-4-yl)methanone (36.4 mg, 0.186 mmol). The

solution was allowed to stir at RT for 24 h. At this time, a 1:1 solution of EtOAc:Et2O (5

mL) was added, and this was washed with aqueous 10% Na2CO3 (3 x 2 mL). The extract

was then dried with anhydrous MgSO4, filtered, and the filtrate was concentrated in

vacuo. The crude residue was purified by column chromatography (20 g silica, 80%

EtOAc/Hex) to provide the title compound. Yield: 35 mg, 0.065 mmol, 35%. 1H NMR

(500 MHz, Chloroform-d) δ 7.70 – 7.64 (m, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.28 (dt, J =

5.9, 1.3 Hz, 1H), 7.17 (td, J = 7.5, 7.0, 1.0 Hz, 1H), 7.12 (dd, J = 8.5, 5.4 Hz, 2H), 6.99 –

6.93 (m, 2H), 6.67 (s, 1H), 5.49 (s, 2H), 4.61 (bs, 1H), 4.24 (bs, J = 8.8, 5.8 Hz, 1H), 3.58

(t, J = 5.5 Hz, 2H), 3.49 – 3.37 (m, 2H), 2.93 (d, J = 12.7 Hz, 2H), 2.75 (dq, J = 14.3, 7.7,

7.1 Hz, 1H), 1.77 – 1.58 (m, 8H), 1.31 – 1.25 (m, 1H), 0.94 – 0.81 (m, 1H). HPLC tR =

7.55 min, >95% purity.

(4-(Dibenzylamino)piperidin-1-yl)(1-(4-fluorobenzyl)-1H-indol-2-yl)methanone

(CCG-204020). Tert-butyl 4-(dibenzylamino)piperidine-1-carboxylate (330 mg, 0.867

mmol) was dissolved in DCM (5 mL), followed by the addition of trifluoroacetic acid

(0.200 mL, 2.60 mmol). The reaction was allowed to stir for 18 h at RT, after which time

160

the solvent was removed in vacuo and the crude residue of N,N-dibenzylpiperidin-4-

amine bis(trifluoroacetate) was used directly in the subsequent reaction

The following reagents were dissolved in anhydrous DMF (5 mL) at RT: 1-(4-

fluorobenzyl)-1H-indole-2-carboxylic acid (233 mg, 0.867 mmol), N,N-

dibenzylpiperidin-4-amine bis(trifluoroacetate) (243 mg, 0.867 mmol), DIPEA (757 µL,

4.34 mmol), EDCHCl (183 mg, 0.954 mmol), and HOBT (146 mg, 0.954 mmol). The

reaction was allowed to stir for 36 h at RT, after which time it was diluted with 1:1

EtOAc:Et2O and washed with 10% aq. Na2CO3 (2X), then dried (MgSO4) and

concentrated. The residue was triturated with Et2O to yield a light brown solid. Yield:

380 mg, 0.715 mmol, 82%. 1H NMR (500 MHz, Chloroform-d) δ 7.69 (dd, J = 8.0, 3.9

Hz, 1H), 7.43 – 7.38 (m, 5H), 7.37 – 7.31 (m, 5H), 7.29 – 7.25 (m, 2H), 7.21 (t, J = 7.3,

3.4 Hz, 1H), 7.11 – 7.07 (m, 2H), 6.94 (t, J = 11.1, 8.7, 2.0 Hz, 2H), 6.65 (s, 1H), 5.53 (s,

2H), 4.78 (bs, 1H), 4.17 (bs, 1H), 3.59 (s, 4H), 2.85 – 2.67 (m, 2H), 2.58 (bs, 1H), 1.92

(bs, 1H), 1.67 (bs, 1H), 1.49 (bs, 1H), 1.04 (bs, J = 30.5 Hz, 1H). HPLC tR = 6.48 min,

>95%.

N-Benzyl-1-(1-(4-chlorobenzyl)-1H-pyrrole-2-carbonyl)piperidine-4-

carboxamide (CCG-204054). The following was dissolved in anhydrous DMF (3 mL): 1-

(1-(4-chlorobenzyl)-1H-pyrrole-2-carbonyl)piperidine-4-carboxylic acid (115 mg, 0.332

mmol), Hunig's Base (0.174 ml, 0.995 mmol), EDCHCl (76 mg, 0.398 mmol), HOBt

(60.9 mg, 0.398 mmol), and benzylamine (0.040 ml, 0.365 mmol). This was stirred at

room temperature for 1 day with 3Å molecular sieves. At this time, a 1:1 solution of

EtOAc:Et2O (20 mL) was added and the organic layer was washed with 10% aq.

161

Na2CO3 (2 x 10 mL) and brine (1 x 10 mL). The organic solution was then dried

(anhydrous MgSO4) and concentrated in vacuo. Trituration in Et2O provided 101 mg of

the title compound as a fine, ruddy brown solid. 1H NMR (500 MHz, Chloroform-d) δ

7.39 – 7.34 (m, 2H), 7.33 – 7.26 (m, 5H), 7.06 (dd, J = 8.9, 2.5 Hz, 2H), 6.82 (dd, J = 2.7,

1.7 Hz, 1H), 6.35 (dd, J = 3.7, 1.6 Hz, 1H), 6.15 (dd, J = 3.7, 2.7 Hz, 1H), 5.76 (s, 1H),

5.30 (s, 2H), 4.47 (d, J = 5.6 Hz, 2H), 4.39 (bd, J = 13.3 Hz, 2H), 2.86 (t, J = 12.7 Hz,

2H), 2.33 (tt, J = 11.3, 3.8 Hz, 1H), 1.81 (d, J = 13.0 Hz, 2H), 1.51 (d, J = 12.9 Hz, 2H).

HPLC tR = 7.06 min, > 95%.

(1-(4-Fluorobenzyl)-1H-indol-2-yl)(4-(piperidin-1-ylmethyl)piperidin-1-

yl)methanone (CCG-204055). Tert-butyl 4-(piperidin-1-ylmethyl)piperidine-1-

carboxylate (100 mg, 0.469 mmol) was dissolved in DCM (5 mL), and TFA (1 mL) was

added. The reaction was allowed to stir for 12 h, after which time the solvent was

stripped off in vacuo, then thoroughly removed under high vacuum. The crude residue

was then taken directly into the next step.

The following was added to anhydrous DMF (2 mL) sequentially: 1-(4-

fluorobenzyl)-1H-indole-2-carboxylic acid (66.8 mg, 0.248 mmol), DIPEA (0.217 ml,

1.240 mmol), EDCHCl (52.3 mg, 0.273 mmol), HOBT (41.8 mg, 0.273 mmol), and 1-

(piperidin-4-ylmethyl)piperidine trifluoroacetate (45.2 mg, 0.248 mmol). This was stirred

at room temperature for 24 h with 3Å MS. At this time, a 1:1 solution of EtOAc:Et2O

was added and the solution was washed with 10% aq. Na2CO3. The organic phase was

dried with MgSO4 and concentrated in vacuo. The residue was then purified by silica gel

162

chromatography (20g silica, 80% EtOAc/Hexanes) to give an oil. Yield: 58 mg , 0.134

mmol, 54%. HPLC tR = 5.36 min, > 95%. 1H NMR (500 MHz, Chloroform-d) δ 7.66 (d,

J = 7.9 Hz, 1H), 7.40 (d, J = 8.3 Hz, 1H), 7.29 (ddd, J = 8.3, 6.9, 1.2 Hz, 1H), 7.18 (ddd,

J = 8.0, 7.0, 0.9 Hz, 1H), 7.14 – 7.09 (m, 2H), 6.99 – 6.92 (m, 2H), 6.63 (d, J = 0.7 Hz,

1H), 5.50 (s, 2H), 4.66 (bs, 1H), 4.10 (bs, 1H), 2.77 (d, J = 64.4 Hz, 2H), 2.41 – 2.27 (m,

4H), 2.11 – 2.02 (m, 2H), 1.83 – 1.53 (m, 5H), 1.48 – 1.39 (m, 2H), 1.05 (bs, 1H), 0.66

(bs, 1H). HPLC tR = 5.36 min, >95% purity.

(1-(4-Fluorobenzyl)-1H-indol-2-yl)(4-(hydroxy(phenyl)methyl)piperidin-1-

yl)methanone (CCG-204056). The following was added sequentially to anhydrous DMF

(5 mL): 1-(4-fluorobenzyl)-1H-indole-2-carboxylic acid (0.119 g, 0.443 mmol),

Hunig'sBase (0.232 ml, 1.329 mmol), EDCHCl (0.093 g, 0.487 mmol), HOBT (0.075 g,

0.487 mmol), and phenyl(piperidin-4-yl)methanol (0.085 g, 0.443 mmol). The solution

was stirred at room temperature for 30 h, at which time a 1:1 solution of EtOAc:Et2O

was added and washed with 10% aq. Na2CO3. The organic solution was dried with

MgSO4 and concentrated in vacuo to give a residue that was purified by silica gel

chromatography (45 g silica, 80% EtOAc/Hex). Yield: 67 mg, . 1H NMR (500 MHz,

Chloroform-d) δ 7.66 (d, J = 7.9 Hz, 1H), 7.38 (dd, J = 14.5, 7.8 Hz, 3H), 7.32 – 7.26 (m,

4H), 7.17 (t, J = 7.5 Hz, 1H), 7.09 (dd, J = 8.5, 5.3 Hz, 2H), 6.98 – 6.92 (m, 2H), 6.61 (s,

1H), 5.48 (d, J = 6.5 Hz, 2H), 4.67 (d, J = 54.5 Hz, 1H), 4.30 – 4.23 (m, 1H), 4.22 – 3.93

(m, 1H), 2.86 – 2.52 (m, 2H), 2.43 – 1.83 (m, 2H), 1.79 (dtd, J = 11.5, 7.7, 3.7 Hz, 1H),

1.28 – 0.94 (m, 2H). HPLC tR = 7.85 min, > 95%.

163

(4-(Benzylamino)piperidin-1-yl)(1-(4-fluorobenzyl)-1H-indol-2-yl)methanone

(CCG-205420). (4-(Dibenzylamino)piperidin-1-yl)(1-(4-fluorobenzyl)-1H-indol-2-

yl)methanone CCG-204020 was dissolved in EtOH (10 mL) and THF (10 mL), along

with a few drops of conc. HCl. This solution was sparged with N2 and Pd(OH)2 was

added. The reaction was then shaken on a hydrogenator at 45 psi H2 at RT for 1 h (longer

reaction times cause rapid accumulation of complete debenzylation.) The reaction was

filtered over celite and the filtrate was diluted with EtOAc and washed with sat. aq.

Na2CO3 (1X), dried (MgSO4) and concentrated to give the desired compound as an oil.

Yield: 77%. 1H NMR (500 MHz, Chloroform-d) δ 7.70 – 7.65 (m, 1H), 7.41 – 7.32 (m,

5H), 7.32 – 7.27 (m, 2H), 7.22 – 7.16 (m, 1H), 7.12 (dd, J = 8.3, 5.3 Hz, 2H), 6.96 (ddd, J

= 9.7, 7.5, 1.1 Hz, 2H), 6.65 (s, 1H), 5.51 (s, 2H), 4.49 (bs, 1H), 4.07 (bs, 1H), 3.83 (s,

2H), 3.00 (bs, 2H), 2.77 (tt, J = 9.9, 4.0 Hz, 1H), 2.04 – 1.64 (m, 2H), 1.32 (m, 2H). TOF

ES+ MS: 442.1 (M + H), 464.1 (M + Na). HPLC tR = 5.83 min, > 90%.

(4-(Cyclohexyl(hydroxy)methyl)piperidin-1-yl)(1-(4-fluorobenzyl)-1H-indol-2-

yl)methanone (CCG-205421). Tert-butyl 4-(cyclohexyl(hydroxy)methyl)piperidine-1-

carboxylate (100 mg, 0.336 mmol) was dissolved in anhydrous THF (5 mL) and TFA (52

µL, 0.672 mmol) and stirred at RT for 15 h. After this time, the solvent was removed in

vacuo via rotovap, then thoroughly removed under high vacuum. The resulting residue

was taken directly into the next step.

4-(Cyclohexyl(hydroxy)methyl)piperidine trifluoroacetate (~105 mg, 0.336

mmol) was dissolved in anhydrous DMF (3 mL), followed by 1-(4-fluorobenzyl)-1H-

indole-2-carboxylic acid (100 mg, 0.370 mmol), DIPEA (0.352 mL, 2.016 mmol),

164


allowed to stir at RT for 24 h, after which time the reaction was diluted with 1:1

EtOAc:Et2O and washed with 10% aq. Na2CO3 (3X), dried (MgSO4), and concentrated.

The resulting residue was purified by silica flash chromatography (20 g silica, 80%

EtOAc:Hex). Yield: 90 mg, 0.202 mmol, 60%. 1H NMR (500 MHz, Chloroform-d) δ

7.66 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 8.3 Hz, 1H), 7.31 – 7.27 (m, 1H), 7.18 (ddd, J = 7.9,

7.0, 1.0 Hz, 1H), 7.14 – 7.08 (m, 2H), 6.98 – 6.92 (m, 2H), 6.64 (d, J = 0.8 Hz, 1H), 5.51

(s, 2H), 4.75 (bs, 1H), 4.15 (bs, 1H), 3.05 (d, J = 5.6 Hz, 1H), 2.83 (bs, 1H), 2.68 (bs,

1H), 1.82 – 1.58 (m, 7H), 1.41 – 1.04 (m, 9H). ES+ TOF MS: 449.2 (M + H), 471.2 (M +

Na). HPLC tR = 8.72 min, > 90%.

N-Benzyl-1-(1-(4-chlorobenzyl)-3-methyl-1H-indole-2-carbonyl)piperidine-4-

carboxamide (CCG-205431). Ethyl 1-(4-chlorobenzyl)-3-methyl-1H-indole-2-

carboxylate (310 mg, 0.946 mmol) was dissolved in THF (20 mL) and 5 M aq. NaOH (20

mL) and stirred at 55 °C for 18 h. The reaction was then cooled to 0 °C and acidified to

pH<1 with conc. HCl and material was extracted from the aqueous phase with 1:1

EtOAc:Et2O. The extract was dried (MgSO4) and concentrated, giving 1-(4-

chlorobenzyl)-3-methyl-1H-indole-2-carboxylic acid as a yellow solid. Yield: 273 mg,

0.911 mmol, 96%.

The following reagents were dissolved in anhydrous DMF (3 mL) at RT: 1-(4-

chlorobenzyl)-3-methyl-1H-indole-2-carboxylic acid (90 mg, 0.315 mmol), N-

benzylpiperidine-4-carboxamide hydrochloride (70 mg, 0.315 mmol), DIPEA (170 µL,

0.945 mmol), and HATU (144 mg, 0.378 mmol). The reaction was allowed to stir for 20

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h at RT, after which time it was diluted with 1:1 EtOAc:Et2O and washed with 10% aq.

Na2CO3 (3X), then dried (MgSO4) and concentrated. The residue was purified by silica

flash chromatography (20 g silica, 50% EtOAc:Hex). Yield: 77 mg, 0.153 mmol, 51%.


7.18 (t, J = 7.8 Hz, 3H), 7.03 (t, J = 8.2 Hz, 2H), 5.73 (s, 1H), 5.57 (s, 1H), 5.38 (d, J =

11.8 Hz, 2H), 4.77 (d, J = 10.9 Hz, 1H), 4.44 (d, J = 5.9 Hz, 2H), 3.76 (d, J = 14.8 Hz,

1H), 3.08 (d, J = 17.3 Hz, 1H), 2.80 (s, 3H), 2.29 (d, J = 21.5 Hz, 3H), 1.96 (d, J = 11.2

Hz, 1H), 1.76 (d, J = 17.2 Hz, 1H), 1.52 (s, 1H). ES+ TOF MS: 500.3 (M + H), 522.2 (M

+ Na). HPLC tR = 7.95 min, > 95%.

(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-(isoindoline-2-carbonyl)piperidin-1-

yl)methanone. (CCG-205470) The following was added sequentially to DCM (2 mL): 1-

(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)piperidine-4-carboxylic acid (50 mg, 0.126

mmol), DIPEA (0.07 mL, 0.378 mmol), EDCHCl (30 mg, 0.151 mmol), HOBT (24 mg,

0.151 mmol), and isoindoline (0.018 mL, 0.151 mmol). The mixture was stirred for 18 h

at RT, at which time the solution diluted with a 1:1 solution of EtOAc:Et2O and washed

with 1M HCl (1X), 10% aq. Na2CO3 (1X) and brine (1X). The organic phase was dried

with magnesium sulfate and concentrated in vacuo. The resulting residue was dissolved

in EtOAc and precipitated out with Hex, and then collected over a filter and washed with

a 10:1 solution of Hex:EtOAc. Yield: 22 mg, 0.044 mmol, 35%. 1H NMR (400 MHz,

Chloroform-d) δ 7.65 (d, J = 7.9 Hz, 1H), 7.40 – 7.21 (m, 8H), 7.15 (t, J = 7.4 Hz, 1H),

7.05 (d, J = 8.3 Hz, 2H), 6.68 (d, J = 1.6 Hz, 1H), 5.47 (s, 2H), 4.88 (s, 2H), 4.81 (s, 2H),

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4.42 (bs, 4H), 2.97 (bs, 2H), 2.72 (td, J = 14.7, 12.7, 4.7 Hz, 1H), 1.78 (bs, 2H). ES+

TOF MS: 498.2 (M + H), 520.2 (M + Na). HPLC tR = 5.85 min, > 95%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(2,3-dihydro-1H-inden-2-yl)piperidine-

4-carboxamide. (CCG-205471) The following was added sequentially to DCM (2 mL):

1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)piperidine-4-carboxylic acid (50 mg, 0.126


0.151 mmol), and 2-aminoindane (0.020 mL, 0.151 mmol). The mixture was stirred for

36 h at rt, at which time the solution diluted with a 1:1 solution of EtOAc:Et2O and

washed with 1M HCl (1X), 10% aq. Na2CO3 (1X) and brine (1X), which resulted in a

precipitate that failed to go into either phase. The precipitate was collected and washed

with water and Et2O to afford the title compound as a white solid. Yield: 52 mg, 0.102


(m, 1H), 7.25 – 7.11 (m, 8H), 7.03 (d, J = 8.1 Hz, 2H), 6.64 (s, 1H), 5.62 (d, J = 7.6 Hz,

1H), 5.46 (s, 2H), 4.83 – 4.69 (m, 1H), 4.32 (bs, 4H), 3.33 (dd, J = 16.3, 7.0 Hz, 2H),

2.89 – 2.72 (m, 4H), 2.27 – 2.14 (m, 1H), 1.74 (bs, 2H). ES+ TOF MS: 512.2 (M + H),

534.2 (M + Na). HPLC tR = 5.96 min, > 95%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(2,3-dihydro-1H-inden-1-

yl)piperidine-4-carboxamide (CCG-205472). The following was added sequentially to

DCM (2 mL): 1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)piperidine-4-carboxylic acid

71 (50 mg, 0.13 mmol), DIPEA (0.07 mL, 0.38 mmol), EDCHCl (30 mg, 0.15 mmol),

HOBT (24 mg, 0.15 mmol), and 1-aminoindane (20 µL, 0.15 mmol). The mixture was

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stirred for 18 h at rt, at which time the solution diluted with a 2:1 solution of EtOAc:Et2O

and washed with 1M HCl (2X), 10% aq. Na2CO3 (1X) and brine (1X). The organic phase

was dried with MgSO4 and concentrated in vacuo. The resulting residue was dissolved in

EtOAc and precipitated out Hex, and then collected over a filter and washed with Et2O to

give the title compound as a white solid. Yield: 33 mg, 0.06 mmol, 51%. 1H NMR (500

MHz, Chloroform-d) δ 7.66 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 8.3 Hz, 1H), 7.29 – 7.20 (m,

7H), 7.17 (t, J = 7.4 Hz, 1H), 7.04 (d, J = 8.0 Hz, 2H), 6.66 (s, 1H), 5.66 (d, J = 8.4 Hz,

1H), 5.47 (s, 2H), 4.49 (bs, 1H), 4.33 (bs, 1H), 3.04 – 2.95 (m, 1H), 2.89 (dt, J = 15.9, 8.0

Hz, 3H), 2.62 (dtd, J = 12.2, 7.8, 3.8 Hz, 1H), 2.33 (tt, J = 11.1, 3.9 Hz, 1H), 1.94 – 1.54

(m, 6H). ES+ TOF MS: 512.2 (M + H), 534.2 (M + Na). HPLC tR = 5.93 min, > 95%.

(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-(2-phenylpyrrolidine-1-carbonyl)piperidin-

1-yl)methanone. (CCG-205473) The following was added sequentially to DCM (2 mL):

1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)piperidine-4-carboxylic acid 71 (50 mg,

0.126 mmol), DIPEA (0.07 mL, 0.378 mmol), EDCHCl (30 mg, 0.151 mmol), HOBT

(24 mg, 0.151 mmol), and 2-phenylpyrrolidine (0.023 mL, 0.151 mmol). The mixture

was stirred for 18 h at rt, at which time the solution diluted with a 1:1 solution of

EtOAc:Et2O and washed with 1M HCl (1X), 10% aq. sodium carbonate (1X) and brine

(1X). The organic phase was dried with magnesium sulfate and concentrated in vacuo.

The resulting residue was dissolved in EtOAc and precipitated out hexanes, and then

collected over a filter and washed with diethyl ether to give the title compound as a white

solid. Yield: 20 mg, 0.038 mmol, 30%. 1H NMR (400 MHz, Chloroform-d) δ 7.66 – 7.57

(m, 1H), 7.33 – 7.14 (m, 10H), 7.00 (d, J = 8.1 Hz, 2H), 6.58 (s, 1H), 5.41 (s, 2H), 4.34

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(s, 4H), 3.73 (dd, J = 19.2, 11.3 Hz, 2H), 3.07 – 2.88 (m, 1H), 2.73 (s, 1H), 2.51 – 2.26

(m, 3H), 2.04 – 1.44 (m, 5H). ES+ TOF MS: 526.2 (M + H), 548.2 (M + Na). HPLC tR =

6.62 min, > 95%.

(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-(3-phenylpyrrolidine-1-carbonyl)piperidin-

1-yl)methanone. (CCG-205474) The following was added sequentially to DCM (2 mL):

1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)piperidine-4-carboxylic acid (50 mg, 0.126


0.151 mmol), and 3-phenylpyrrolidine (0.023 mL, 0.151 mmol). The mixture was stirred

for 36 h at rt, at which time the solution diluted with a 1:1 solution of EtOAc:Et2O and

washed with 1M HCl (1X), 10% aq. sodium carbonate (1X) and brine (1X). The organic

phase was dried with magnesium sulfate and concentrated in vacuo. The resulting residue

was dissolved in EtOAc and precipitated out hexanes, and then collected over a filter and

washed with diethyl ether to give the title compound as a slightly yellow solid. Yield: 21

mg, 0.040 mmol, 32%. 1H NMR (500 MHz, Chloroform-d) δ 7.66 (dd, J = 7.8, 2.8 Hz,

1H), 7.34 (dq, J = 14.5, 7.4 Hz, 3H), 7.29 – 7.20 (m, 6H), 7.16 (t, J = 7.8 Hz, 1H), 7.06

(dd, J = 7.9, 5.4 Hz, 2H), 6.68 (d, J = 6.3 Hz, 1H), 5.48 (d, J = 5.0 Hz, 2H), 4.57 (s, 1H),

4.33 (s, 1H), 4.03 (dd, J = 11.9, 7.5 Hz, 1H), 3.98 – 3.91 (m, 1H), 3.85 (ddd, J = 11.6,

8.3, 2.4 Hz, 1H), 3.73 (td, J = 9.1, 8.3, 2.9 Hz, 1H), 3.60 (td, J = 9.6, 6.8 Hz, 1H), 3.49

(ddd, J = 21.3, 9.6, 4.0 Hz, 2H), 3.38 (p, J = 7.9 Hz, 1H), 2.92 (s, 2H), 2.62 (dp, J = 21.4,

7.5 Hz, 1H), 2.46 – 2.36 (m, 1H), 2.31 (dq, J = 13.1, 4.1, 2.6 Hz, 1H), 2.16 – 2.00 (m,

2H). ES+ TOF MS: 526.3 (M + H), 548.2 (M + Na). HPLC tR = 6.37 min, > 95%.

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(4-(Cyclohexanecarbonyl)piperidin-1-yl)(1-(4-fluorobenzyl)-1H-indol-2-yl)methanone

(CCG-205475). 4-(Cyclohexyl(hydroxy)methyl)piperidin-1-yl)(1-(4-fluorobenzyl)-1H-

indol-2-yl)methanone (85 mg, 0.189 mmol) was dissolved in DCM (4 mL) and cooled to

-78 °C. Anhydrous DMSO (34 µL, 0.474 mmol) and (COCl)2 (34 µL, 0.379 mmol) were

added, and the solution was stirred for 30 min, at which time TEA (130 µL, 0.947 mmol)

was added dropwise and the reaction was allowed to stir for an additional 30 min, then

the bath was removed and it was allowed to stir until it reached RT. The reaction was

diluted with 1:1 EtOAc:Et2O and washed with H2O (2X) and brine (1X), dried (MgSO4),

and concentrated in vacuo. The residue was purified via silica flash chromatography (50

g silica, 10% EtOAc:Hex). Yield: 49 mg, 0.106 mmol, 58%. 1H NMR (500 MHz,


7.15 (t, J = 7.5 Hz, 1H), 7.08 (dd, J = 8.2, 5.5 Hz, 2H), 6.99 – 6.89 (m, 2H), 6.62 (s, 1H),

5.47 (s, 2H), 4.54 (bs, 1H), 4.15 (bs, 1H), 2.94 – 2.81 (m, 2H), 2.68 (tt, J = 11.2, 3.6 Hz,

1H), 2.46 (tt, J = 11.1, 2.9 Hz, 1H), 1.82 – 1.64 (m, 7H), 1.38 – 1.17 (m, 7H). TOF ES+

MS: (M + Na) 469.2. HPLC tR = 8.93 min, > 90%.

(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-(piperidin-1-ylmethyl)piperidin-1-

yl)methanone (CCG-205476). Tert-Butyl 4-(piperidin-1-ylmethyl)piperidine-1-

carboxylate (238 mg, 0.843 mmol) was dissolved in DCM (10 mL), and TFA (2 mL) was

added. The reaction was allowed to stir for 12 h, after which time the solvent was

stripped off in vacuo, then thoroughly removed under high vacuum. The crude residue

was then taken directly into the next step.

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The following was added to DCM (6 mL): 1-(4-chlorobenzyl)-1H-indole-2-

carboxylic acid (110 mg, 0.380 mmol), DIPEA (0.50 mL, 2.95 mmol), EDCHCl (100

mg, 0.506 mmol), HOBT (80 mg, 0.506 mmol), and 1-(piperidin-4-ylmethyl)piperidine

(150 mg, 0.843 mmol). The solution was stirred at RT for 15 h, at which time the solution

was diluted with a 1:1 solution of EtOAc:Et2O and washed with H2O (1X), 10% aq.

Na2CO3 (1X), and brine (1X). The organic phase was then dried over MgSO4 and

concentrated in vacuo. The resulting residue was purified via silica flash chromatography

(40 g silica, 10% EtOAc:Hex) and triturated with EtOAc and Hex to afford the title

compound as a white powder. Yield: 109 mg, 0.242 mmol, 63%. 1H NMR (400 MHz,


7.03 (d, J = 8.4 Hz, 2H), 6.59 (s, 1H), 5.46 (s, 2H), 4.59 (bs, 2H), 4.09 (bs, 2H), 2.73 (bs,

2H), 2.34 (bs, 2H), 2.08 (d, J = 6.8 Hz, 2H), 1.67 (s, 6H), 1.44 – 1.32 (m, 2H), 0.95 (bs,

1H), 0.56 (bs, 1H). ES+ TOF MS: 450.2 (M + H). HPLC tR = 6.18 min, > 95%.

(4-Benzylpiperazin-1-yl)(1-(4-chlorobenzyl)-1H-indol-2-yl)methanone (CCG-

206327). The following was added to DCM (5 mL): 1-(4-chlorobenzyl)-1H-indole-2-


mg, 0.294 mmol), HOBT (45 mg, 0.294 mmol), and 1-benzylpiperazine (0.085 mL,

0.490 mmol). The solution was stirred at RT for 16 h, at which time the solution was

diluted with a 1:1 solution of EtOAc:Et2O and washed with H2O (1X), 10% aq. Na2CO3

(1 x), and brine (1 x). The organic phase was then dried over MgSO4 and concentrated in

vacuo. The resulting residue was purified via silica flash chromatography (30 g silica,

10% EtOAc:Hex) and triturated with EtOAc and Hex to afford the title compound as a

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white powder. Yield: 52 mg, 0.117 mmol, 48%. 1H NMR (400 MHz, Chloroform-d) δ

7.62 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.32 – 7.19 (m, 8H), 7.14 (t, J = 7.5

Hz, 1H), 7.02 (d, J = 8.2 Hz, 2H), 6.60 (s, 1H), 5.48 (s, 2H), 3.63 (bs, 4H), 3.42 (s, 2H),

2.35 (bs, 2H), 2.04 (bs, J = 11.7 Hz, 2H). TOF ES+ MS: (M + H) 444.2, (M + Na) 466.2.

HPLC tR = 6.12 min, > 95%.

(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-(indoline-1-carbonyl)piperidin-1-

yl)methanone. (CCG-206328) The following was added sequentially to DCM (5 mL): 1-

(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)piperidine-4-carboxylic acid (100 mg, 0.252


0.302 mmol), and indoline (0.040 mL, 0.302 mmol). The mixture was stirred for 24 h at

rt, at which time the solution diluted with EtOAc and washed with water (2X) and brine

(1X). The organic phase was dried with magnesium sulfate and concentrated in vacuo.

The resulting residue was dissolved in EtOAc and precipitated out hexanes, and then

collected over a filter and washed with diethyl ether to give the title compound as a white

solid. Yield: 1H NMR (400 MHz, Chloroform-d) δ 7.66 (d, J = 7.8 Hz, 1H), 7.33 (t, J =

8.8 Hz, 1H), 7.30 – 7.13 (m, 8H), 7.04 (d, J = 8.2 Hz, 2H), 6.65 (s, 1H), 5.48 (s, 2H),

4.36 (s, 2H), 3.39 (t, J = 5.8 Hz, 2H), 3.08 (q, J = 9.6, 9.1 Hz, 2H), 2.99 – 2.82 (m, 2H),

2.71 – 2.64 (m, 2H), 2.26 (tt, J = 11.3, 3.9 Hz, 1H), 1.76 (s, 2H), 1.50 (s, 2H). TOF ES+

MS: (M + H) 498.2. HPLC tR = 8.58 min, >95% purity.

1-(1-(4-Chlorobenzyl)-1H-pyrrole-2-carbonyl)-N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (CCG-206381). The following was added

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sequentially to DCM: 1-(1-(4-chlorobenzyl)-1H-pyrrole-2-carbonyl)piperidine-4-

carboxylic acid (600 mg, 1.73 mmol), TEA (0.725 mL, 5.19 mmol), EDCHCl (365 mg,

1.903 mmol), and HOBt (291 mqg, 1.903 mmol). This was allowed to stir at rt for 30

min, at which time pyridylethylamine ( 0.227 mL, 1.903 mmol) was added. Stirring

continued for 18 h. At this time, the DCM and TEA were stripped off, and the residue

was taken up in EtOAc and washed with 10% aqueous sodium carbonate (3X). The

organic phase was collected, dried over magnesium sulfate, and concentrated in vacuo.

The resulting solid/oil mixture was recrystallized from EtOAc to afford the title

compound as small, white crystals. Yield: 586 mg, 1.29 mmol, 75%. 1HNMR (400 MHz,

CDCl3) δ 8.50 (d, J = 5.7 Hz, 2H), 7.21 (d, J = 8.3 Hz, 2H), 7.09 (d, J = 5.6 Hz, 2H), 7.02

(d, J = 8.2 Hz, 2H), 6.78 (s, 1H), 6.30 (dd, J = 3.6, 1.2 Hz, 1H), 6.13−6.09 (m, 1H), 5.48

(t, J = 5.4 Hz, 1H), 5.26 (s, 2H), 4.32 (d, J = 12.7 Hz, 2H), 3.53 (q, J = 6.8 Hz, 2H), 2.82

(t, J = 7.0 Hz, 4H), 2.19 (tt, J = 11.2, 3.6 Hz, 1H), 1.68 (d, J = 14.4 Hz, 2H), 1.35 (q, J =

11.0, 9.8 Hz, 2H). Anal. Calcd for C25H27ClN4O2: C, 66.58%; H, 6.04%; N, 12.42%.

Found: C, 66.68%; H, 6.32%; N, 12.38%. TOF ES+ MS: (M + H) 451.2, (M + Na) 473.2.

HPLC tR = 5.03 min, >95% purity.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-((2,3-dihydro-1H-inden-2-

yl)methyl)piperidine-4-carboxamide (CCG-206382). The following was added

sequentially to DCM (8 mL): 1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)piperidine-4-


mg, 0.499 mmol), HOBT (76 mg, 0.499 mmol), and crude (2,3-dihydro-1H-inden-2-

yl)methanamine (105 mg, 0.713 mmol). The mixture was stirred for 18 h at RT, at which

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time the solution diluted with a 1:1 solution of EtOAc:Et2O and washed with 1 M HCl

(1X), 10% aq. Na2CO3 (1X). The organic phase was dried with MgSO4 and concentrated

in vacuo. Purification was accomplished via silica flash chromatography (60 g silica,

50% EtOAc:Hex), followed by EtOAc/Hex tritutation to give the title compound as a

white powder. Yield: 137 mg, 0.260 mmol, 57%. 1H NMR (400 MHz, Chloroform-d) δ

7.66 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 8.8 Hz, 1H), 7.30 – 7.13 (m, 8H), 7.04 (d, J = 8.2

Hz, 2H), 6.65 (s, 1H), 5.48 (s, 2H), 4.36 (s, 2H), 3.39 (t, J = 5.8 Hz, 2H), 3.08 (q, J = 9.6,

9.1 Hz, 2H), 2.99 – 2.82 (m, 2H), 2.71 – 2.64 (m, 2H), 2.26 (tt, J = 11.3, 3.9 Hz, 1H),

1.76 (s, 2H), 1.50 (s, 2H). TOF ES+ MS: (M + H) 526.2, (M + Na) 548.2. HPLC tR =

8.25 min, > 95%.

N-Benzyl-1-(1-(4-chlorobenzyl)-7-methyl-1H-indole-2-carbonyl)piperidine-4-

carboxamide (CCG-206447). The following was added to sequentially to anhydrous

DMF (2 mL): 1-(4-chlorobenzyl)-7-methyl-1H-indole-2-carboxylic acid (50 mg, 0.167

mmol), DIPEA (0.10 mL, 0.584 mmol), EDCHCl (38 mg, 0.200 mmol), and HOBT (31

mg, 0.200 mmol). This was allowed to stir for 30 min at RT, after which time the crude

N-benzylpiperidine-4-carboxamide (36 mg, 0.20 mmol) was added. The reaction was

stirred at RT for 18 h, at which time the reaction was diluted with 1:1 EtOAc:Et2O and

washed with 10% aq. Na2CO3 (2x), dried over MgSO4, and concentrated. The residue

was purified via silica flash chromatography (20 g silica, 50% EtOAc:Hex) to afford the

title compound as a white powder. Yield: 39 mg, 0.078 mmol, 47%. 1H NMR (500 MHz,

Chloroform-d) δ 7.51 (d, J = 7.7 Hz, 1H), 7.37 – 7.33 (m, 2H), 7.31 – 7.26 (m, 3H), 7.19

(dd, J = 10.0, 3.6 Hz, 2H), 7.08 – 6.99 (m, 2H), 6.82 (d, J = 8.2 Hz, 2H), 6.64 (s, 1H),

174

5.73 (s, 2H), 4.58 (bs, 1H), 4.45 (t, J = 5.9 Hz, 2H), 4.11 (bs, 1H), 2.83 (bs, 2H), 2.58 (s,

3H), 2.34 – 2.27 (m, 1H), 1.80 (bs, 2H), 1.37 (bs, 2H). TOF ES+ MS: (M + H) 500.2, ( M

+ Na) 522.2. HPLC tR = 8.01 min, > 95%.

(1-(4-Fluorobenzyl)-1H-indol-2-yl)(4-(hydroxymethyl)piperidin-1-yl)methanone

(CCG-206462). The following was added sequentially to DMF (10 mL): 1-(4-

fluorobenzyl)-1H-indole-2-carboxylic acid (1.175 g, 4.36 mmol), DIPEA (2.29 mL,

13.09 mmol), EDCHCl (1.00 g, 5.24 mmol), and HOBT (802 mg, 5.24 mmol). This

solution was allowed to stir at RT for 1.5 h, at which time 4-piperidinemethanol (754 mg,

6.55 mmol) was added. Stirring continued for 22 h at RT, at which time the solution was

partitioned between H2O and 1:1 solution of EtOAc:Et2O. The organic extract was then

washed with saturated aq. Na2CO3, dried with MgSO4, and concentrated in vacuo.

Purification was accomplished via silica flash chromatography (100 g silica, 80%

EtOAc/Hexanes.) Resulting residue was crystallized via Et2O:Hex trituration to give the

title compound as a white solid. Yield: 1.51 g, 4.11 mmol, 94%. 1H NMR (400 MHz,


7.14 (t, J = 7.5 Hz, 1H), 7.10 – 7.04 (m, 2H), 6.91 (t, J = 8.2 Hz, 2H), 6.60 (s, 1H), 5.46

(s, 2H), 4.64 (bs, 1H), 4.14 (bs, 1H), 3.42 (t, J = 5.1 Hz, 2H), 2.76 (s, 2H), 1.44 (t, J = 5.0

Hz, 1H), 1.05 (bs, 1H), 0.71 (bs, 1H). HPLC tR = 6.62 min, >95% purity.

2-Benzyl-5-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)octahydro-1H-pyrrolo[3,4-

c]pyridin-1-one. (CCG-206485) 5-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)octahydro-

1H-pyrrolo[3,4-c]pyridin-1-one (64 mg, 0.245 mmol) was dissolved in anhydrous DMF

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(5 mL). A 60% oil suspension of NaH (29 mg, 0.735 mmol) was added at RT under N2

and stirred for 1 h. BnBr (0.035 mL, 0.294 mmol) was added and stirring continued for

18 h under the same conditions. After this time, the solution was diluted with EtOAc and

washed with 10% aq. Na2CO3 (2X), dried with MgSO4, and concentrated in vacuo.

Purification was done via silica flash chromatography (25 g silica, 80% EtOAc:Hex.) The

residue was dissolved in DCM, and rapid solvent removal afforded the title compound as

a white powder. Yield: 56 mg, 0.112 mmol, 72%. 1H NMR (500 MHz, Chloroform-d) δ

7.71 (d, J = 7.9 Hz, 1H), 7.37 – 7.26 (m, 5H), 7.26 – 7.20 (m, 5H), 7.15 (t, J = 7.4 Hz,

1H), 7.10 (d, J = 8.2 Hz, 2H), 5.49 (d, J = 3.7 Hz, 2H), 4.55 (d, J = 14.6 Hz, 2H), 4.39 (d,

J = 14.5 Hz, 1H), 4.12 (bs, 1H), 3.43 – 3.30 (m, 2H), 2.98 – 2.89 (m, 1H), 2.84 (d, J = 1.6

Hz, 1H), 2.63 – 2.44 (m, 2H), 1.75 (bs, 1H), 1.41 (bs, J = 13.5 Hz, 1H). TOF ES+ MS:

(M + H) 498.2, (M + Na) 520.2. HPLC tR = 8.23 min, >95% purity.

(1-(4-Fluorobenzyl)-1H-indol-2-yl)(4-(((4-methylphenethyl)amino)methyl)-

piperidin-1-yl)methanone (CCG-206486). 1-(1-(4-fluorobenzyl)-1H-indole-2-

carbonyl)piperidine-4-carbaldehyde (46 mg, 0.126 mmol) was dissolved in THF (2 mL)

and 2-(p-tolyl)ethanamine (0.030 mL, 0.189 mmol) was added. The solution was stirred

at rt for 10 h, at which time the THF was removed in vacuo and the residue dissolved in

EtOH (5 mL), and sodium cyanoborohydride (24 mg, 0.349 mmol) and a catalytic drop

of glacial acetic acid were added. Stirring was permitted for 14 h, at which time the

solvent was removed in vacuo, the residue was taken up in EtOAc. The organic phase

was washed with 10% aq. sodium carbonate, dried over magnesium sulfate, and

concentrated. Purification was accomplished via silica gel flash chromatography (30 g

176

silica gel, 5:95 7M methanolic ammonia:ethyl acetate.) The title compound was obtained

as a yellow oil. Yield: 26 mg, 0.054 mmol, 43%. 1H NMR (500 MHz, Chloroform-d) δ

7.64 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.27 (t, J = 7.7 Hz, 2H), 7.18 – 7.13

(m, 1H), 7.12 – 7.06 (m, 5H), 6.91 (t, J = 8.5 Hz, 2H), 6.60 (s, 1H), 5.47 (s, 2H), 4.62 (bs,

1H), 4.09 (bs, 1H), 2.84 (t, J = 7.0 Hz, 2H), 2.76 (t, J = 7.0 Hz, 2H), 2.70 (bs, 2H), 2.44

(d, J = 5.9 Hz, 2H), 2.32 (s, 3H), 1.79 – 1.62 (m, 5H). TOF ES+ MS: (M + H) 484.1, (M

+ Na) 506.1. HPLC tR = 6.43 min, >95% purity.

(R)-(1-(4-Fluorobenzyl)-1H-indol-2-yl)(4-(((1-phenylethyl)amino)methyl)-



and (R)-α-methylbenzylamine (0.048 mL, 0.375 mmol) was added. The solution was

stirred at rt for 10 h, at which time the THF was removed in vacuo and the residue

dissolved in EtOH (5 mL), and sodium cyanoborohydride (47 mg, 0.749 mmol) and a

catalytic drop of glacial acetic acid were added. Stirring was permitted for 15 h, at which

time the solvent was removed in vacuo, the residue was taken up in EtOAc. The organic

phase was washed with 10% aq. sodium carbonate, dried over magnesium sulfate, and


silica gel, 5:95 7M methanolic ammonia:ethyl acetate.) The title compound was obtained

as a yellow oil. Yield: 52 mg, 0.111 mmol, 44%. 1H NMR (500 MHz, Chloroform-d) δ

7.64 (d, J = 7.9 Hz, 1H), 7.45 – 7.25 (m, 7H), 7.16 (t, J = 7.5 Hz, 1H), 7.10 – 7.01 (m,

2H), 6.91 – 6.80 (m, 2H), 6.60 (s, 1H), 5.46 (s, 2H), 4.60 (s, 1H), 4.05 (s, 1H), 3.80 (q, J

177

= 6.3 Hz, 1H), 2.80 (s, 2H), 2.38 (dd, J = 11.6, 6.0 Hz, 1H), 2.23 (dd, J = 16.5, 4.8 Hz,

1H), 1.70 (d, J = 54.9 Hz, 4H), 1.42 (d, J = 6.6 Hz, 3H), 0.99 (s, 1H), 0.57 (s, 1H). TOF

ES+ MS: (M + H) 470.3, (M + Na) 492.2. HPLC tR = 5.96 min, >95% purity.

(4-((Benzylamino)methyl)piperidin-1-yl)(1-(4-fluorobenzyl)-1H-indol-2-

yl)methanone. (CCG-206500). 1-(1-(4-fluorobenzyl)-1H-indole-2-carbonyl)piperidine-4-

carbaldehyde (52 mg, 0.143 mmol) was dissolved in THF (2 mL) and benzylamine

(0.025 mL, 0.285 mmol) was added. The solution was stirred at RT for 10 h, at which

time the THF was removed in vacuo and the residue dissolved in EtOH (5 mL), and

NaCNBH3 (18 mg, 0.285 mmol) and a catalytic drop of glacial AcOH were added.

Stirring was permitted for 14 h, at which time the solvent was removed in vacuo, the

residue was taken up in EtOAc. The organic phase was washed with 10% aq. Na2CO3,

dried MgSO4, and concentrated. Purification was accomplished via silica flash

chromatography (30 g silica gel, 5:95 7M methanolic ammonia:EtOAc.) The title

compound was obtained as a yellow oil. Yield: 23 mg, 0.050 mmol, 40%. 1H NMR (500

MHz, Chloroform-d) δ 7.65 (d, J = 7.9 Hz, 1H), 7.39 – 7.27 (m, 7H), 7.16 (t, J = 7.5 Hz,

1H), 7.09 (dd, J = 8.2, 5.5 Hz, 2H), 6.91 (t, J = 8.6 Hz, 2H), 6.61 (s, 1H), 5.48 (s, 2H),

4.65 (bs, 1H), 4.10 (bs, 1H), 3.78 (s, 2H), 2.77 (bd, J = 50.6 Hz, 2H), 2.46 (d, J = 6.4 Hz,

2H), 1.79 – 1.61 (m, 3H), 1.09 (bs, 1H), 0.66 (bs, 1H). TOF ES+ MS: (M + H) 456.2, (M

+ Na) 478.2. HPLC tR = 6.04 min, >90% purity.

(S)-(1-(4-Fluorobenzyl)-1H-indol-2-yl)(4-(((1-phenylethyl)amino)methyl)-


178


and (S)-α-methylbenzylamine (0.013 mL, 0.115 mmol) was added. The solution was

stirred at RT for 10 h, at which time the THF was removed in vacuo and the residue

dissolved in EtOH (5 mL), and sodium cyanoborohydride (13 mg, 0.192 mmol) and a

catalytic drop of glacial AcOH were added. Stirring was permitted for 14 h, at which time

the solvent was removed in vacuo, the residue was taken up in EtOAc. The organic phase

was washed with 10% aq. Na2CO3, dried over MgSO4, and concentrated. Purification was

accomplished via silica flash chromatography (30 g silica gel, 5:95 7M methanolic

ammonia:EtOAc.) The title compound was obtained as a yellow oil. Yield: 21 mg, 0.045


(m, 7H), 7.16 (t, J = 7.5 Hz, 1H), 7.08 (dd, J = 8.1, 5.5 Hz, 2H), 6.88 (t, J = 7.9 Hz, 2H),

6.59 (s, 1H), 5.47 (s, 2H), 4.61 (s, 1H), 4.05 (s, 1H), 3.74 – 3.69 (m, 1H), 2.75 (d, J =

49.0 Hz, 2H), 2.41 – 2.33 (m, 1H), 2.22 (dd, J = 11.6, 7.1 Hz, 1H), 1.59 (d, J = 10.4 Hz,

4H), 1.35 (d, J = 6.6 Hz, 3H), 1.01 (s, 1H), 0.63 (s, 1H). TOF ES+ MS: (M + H) 470.3,

(M + Na) 492.2. HPLC tR = 5.98 min, > 95% purity.

(1-(4-Fluorobenzyl)-1H-indol-2-yl)(4-(morpholinomethyl)piperidin-1-

yl)methanone. (CCG-206502) 1-(1-(4-fluorobenzyl)-1H-indole-2-carbonyl)piperidine-4-

carbaldehyde (52 mg, 0.143 mmol) was dissolved in EtOH (5 mL) and morpholine (0.025

mL, 0.285 mmol), sodium cyanoborohydride (18 mg, 0.285 mmol), and a catalytic drop

of glacial acetic acid were added. Stirring was permitted for 13 h, at which time the

solvent was removed in vacuo, the residue was taken up in EtOAc. The organic phase

was washed with 10% aq. sodium carbonate, dried over magnesium sulfate, and

179


silica gel, 100% EtOAc.) The title compound was obtained as an oil. Yield: 15 mg, 0.034


8.3 Hz, 1H), 7.30 – 7.27 (m, 1H), 7.17 (t, J = 8.3 Hz, 1H), 7.12 – 7.07 (m, 2H), 6.97 –

6.89 (m, 2H), 6.63 – 6.59 (m, 1H), 5.49 (s, 2H), 4.65 (bs, 1H), 4.11 (bs, 1H), 3.69 (s, 4H),

2.76 (bd, J = 43.7 Hz, 2H), 2.39 (s, 4H), 2.11 (s, 2H), 1.83 – 1.57 (m, 3H), 0.99 (bs, 1H),

0.60 (bs, 1H). TOF ES+ MS: (M + H) 436.2, (M + Na) 458.2. HPLC tR = 5.38 min,

>95% purity.

(4-((Benzyl(methyl)amino)methyl)piperidin-1-yl)(1-(4-fluorobenzyl)-1H-indol-2-

yl)methanone (CCG-206503). 1-(1-(4-fluorobenzyl)-1H-indole-2-carbonyl)piperidine-4-

carbaldehyde (86 mg, 0.236 mmol) was dissolved in EtOH (5 mL) and N-

methylbenzylamine (0.027 mL, 0.283 mmol), NaCNBH3 (30 mg, 0.472 mmol), and a

catalytic drop of glacial AcOH were added. Stirring was permitted for 14 h, at which time

the solvent was removed in vacuo, the residue was taken up in EtOAc. The organic phase

was washed with 10% aq. Na2CO3, dried over MgSO4, and concentrated. Purification was

accomplished via silica flash chromatography (30 g silica gel, 100% EtOAc.) The title

compound was obtained as an oil. Yield: 23 mg, 0.049 mmol, 20%. 1H NMR (500 MHz,

Chloroform-d) δ 7.64 (t, J = 7.4 Hz, 1H), 7.39 (t, J = 7.7 Hz, 1H), 7.35 – 7.22 (m, 6H),

7.16 (q, J = 7.3 Hz, 1H), 7.07 (td, J = 8.0, 5.4 Hz, 2H), 6.87 – 6.79 (m, 2H), 6.59 (d, J =

7.0 Hz, 1H), 5.47 (s, 2H), 4.61 (bs, 1H), 4.06 (bs, 1H), 3.46 (s, 2H), 2.70 (bs, 2H), 2.18

(d, J = 7.2 Hz, 3H), 2.10 – 1.60 (m, 5H), 0.95 (bs, 1H), 0.50 (bs, 1H). TOF ES+ MS: (M

+ H) 470.2, (M + Na) 492.2. HPLC tR = 6.04 min, > 95% purity.

180

(1-(4-Fluorobenzyl)-1H-indol-2-yl)(4-(((2-(pyridin-4-yl)ethyl)amino)methyl)-


carbonyl)piperidine-4-carbaldehyde (100 mg, 0.273 mmol) was dissolved in EtOH (5

mL) and 4-(2-aminoethyl)pyridine (50 µL, 0.410 mmol), NaCNBH3 (51 mg, 0.819

mmol), and a catalytic drop of glacial AcOH were added. Stirring was permitted for 10 h,

at which time the solvent was removed in vacuo and the residue was taken up in EtOAc.

The organic phase was washed with 10% aq. Na2CO3, dried over MgSO4, and

concentrated. Purification was accomplished via silica flash chromatography (30 g silica

gel, 100% EtOAc.) The title compound was obtained as an oil. Yield: 51 mg, 0.109


7.9 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.29 – 7.26 (m, 1H), 7.17 – 7.11 (m, 3H), 7.10 –

7.06 (m, 2H), 6.97 – 6.89 (m, 3H), 6.60 (s, 1H), 5.47 (s, 2H), 4.65 (bs, 1H), 4.08 (bs, 1H),

2.87 (t, J = 7.1 Hz, 2H), 2.78 (t, J = 7.0 Hz, 2H), 2.70 (bs, 2H), 2.43 (s, 2H), 1.72 – 1.54

(m, 5H). TOF ES+ MS: (M + H) 471.3, (M + Na) 493.2. HPLC tR = 5.51 min, >95%

purity.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(1-(2,3-dihydro-1H-inden-2-

yl)ethyl)piperidine-4-carboxamide (CCG-206549). The following was added



mg, 0.330 mmol), HOBT (51 mg, 0.330 mmol), and crude 2-(1-aminoethyl)indane (~44

181

mg, 0.275 mmol). The mixture was stirred for 16 h at rt, at which time the solution

diluted with a 1:1 solution of ethyl acetate:diethyl ether and washed with 1M HCl (1X),

10% aq. sodium carbonate (1X) and brine (1X). The organic phase was dried with

magnesium sulfate and concentrated in vacuo. The resulting residue was dissolved in

EtOAc and precipitated out hexanes, and then collected over a filter and washed with

diethyl ether to give the title compound as a white solid. Yield: 59 mg, 0.109 mmol, 40%.


7.29 – 7.13 (m, 8H), 7.04 (d, J = 8.2 Hz, 2H), 6.65 (s, 1H), 5.48 (s, 2H), 5.30 (d, J = 8.9

Hz, 1H), 4.45 (s, 1H), 4.25 – 4.14 (m, 1H), 3.02 (td, J = 16.7, 8.3 Hz, 2H), 2.91 – 2.65

(m, 4H), 2.51 (h, J = 8.1 Hz, 1H), 2.22 (tq, J = 10.8, 4.1 Hz, 1H), 1.63 (d, J = 95.6 Hz,

5H), 1.21 (d, J = 6.7 Hz, 3H). TOF ES+ MS: (M + H) 540.2. HPLC tR = 8.55 min, >95%

purity.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-((2,3-dihydro-1H-inden-1-

yl)methyl)piperidine-4-carboxamide. (CCG-206550) The following was added



mg, 0.408 mmol), HOBT (62.4 mg, 0.408 mmol), and 2-indanmethanamine

hydrochloride (62 mg, 0.340 mmol). The mixture was stirred for 18 h at rt, at which time

the solution diluted with a 1:1 solution of ethyl acetate:diethyl ether and washed with 1M

HCl (1X), 10% aq. sodium carbonate (1X) and brine (1X). The organic phase was dried

with magnesium sulfate and concentrated in vacuo. The resulting residue was dissolved

in EtOAc and precipitated out hexanes, and then collected over a filter and washed with

182

diethyl ether to give the title compound as a white solid. Yield: 72 mg, 0.137 mmol, 41%.


7.27 – 7.22 (m, 3H), 7.21 – 7.13 (m, 5H), 7.01 (d, J = 8.2 Hz, 2H), 6.62 (s, 1H), 5.45 (s,

2H), 5.38 (t, J = 5.3 Hz, 1H), 4.36 (s, 2H), 3.62 (dt, J = 12.2, 5.8 Hz, 1H), 3.50 – 3.34 (m,

2H), 2.88 (tt, J = 15.8, 8.2 Hz, 4H), 2.23 (ddd, J = 13.3, 9.5, 5.8 Hz, 2H), 1.78 (ddd, J =

13.3, 8.2, 6.4 Hz, 3H), 1.49 (s, 2H). TOF ES+ MS: (M + H) 526.2. HPLC tR = 8.35 min,

>95% purity.

N-Benzyl-1-(1-(4-chlorobenzyl)-1H-imidazole-2-carbonyl)piperidine-4-

carboxamide (CCG-206586). Ethyl 1-(4-chlorobenzyl)-1H-imidazole-2-carboxylate

(7.28 g, 27.5 mmol), was dissolved in EtOH (10 mL) and 10% aq. NaOH (20 mL) and

stirred at rt for 15 h. The solvent was then stripped off, water was added, and the solution

acidified with HCl. The resulting precipitate was collected over a filter and washed with

1M HCl and dried to afford the carboxylic acid as a white powder which was taken

directly into the next step. Yield: 6.506 g, 27.5 mmol, 100%.

The following was added sequentially to DMF: 1-(4-chlorobenzyl)-1H-imidazole-

2-carboxylic acid (71 mg, 0.298 mmol), DIPEA (0.16 mL, 0.894 mmol), EDCHCl (69

mg, 0.358 mmol), and HOBT (55 mg, 0.358 mmol). This was allowed to stir at rt for 30

min, at which time N-benzylpiperidine-4-carboxamide (~ 65 mg, 0.298 mmol) was added

and syirring continued at rt for 14 h. Addition of water caused precipitation, which was

collected over a filter and washed with water and small amount of diethyl ether and

EtOAc to give the title compound as a white solid. Yield: 37 mg, 0.085 mmol, 28%. 1H

NMR (400 MHz, Chloroform-d) δ 7.34 – 7.22 (m, 7H), 7.12 (d, J = 8.3 Hz, 2H), 7.04 (s,

183

1H), 6.93 (s, 1H), 5.72 (t, J = 5.1 Hz, 1H), 5.36 (d, J = 5.1 Hz, 2H), 4.63 (t, J = 15.1 Hz,

2H), 4.43 (d, J = 5.6 Hz, 2H), 3.14 (t, J = 12.6 Hz, 1H), 2.82 (t, J = 12.8 Hz, 1H), 2.38 (tt,

J = 11.3, 3.9 Hz, 1H), 1.95 (d, J = 13.4 Hz, 1H), 1.83 (d, J = 13.3 Hz, 1H), 1.72 – 1.55

(m, 2H). TOF ES+ MS: (M + H) 437.2, (M + Na) 459.2. HPLC tR = 5.59 min, >95%

purity.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(3-phenylpropyl)piperidine-4-

carboxamide (CCG-206587). The following were added sequentially to DCM (10

mL): 1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)piperidine-4-carboxylic acid (100 mg,

0.252 mmol), DIPEA (132 µL, 0.756 mmol), EDCHCl (58 mg, 0.302 mmol), HOBt (46

mg, 0.302 mmol), and then 3-phenylpropylamine (43 µL, 0.302 mmol). The reaction was

allowed to stir at RT for 13 h, at which time it was diluted with EtOAc, washed with 1 M

HCl (3X), H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X), dried over MgSO4, and

concentrated in vacuo. The residue was the purified by silica flash chromatography (20 g

silica, 70% EtOAc:Hex) to provide the desired material as a colorless oil. Yield: 101 mg,

0.197 mmol, 78%. 1H NMR (500 MHz, Chloroform-d) δ 7.65 (d, J = 7.9 Hz, 1H), 7.37 –

7.23 (m, 5H), 7.24 – 7.12 (m, 6H), 6.64 (s, 1H), 5.45 (s, 2H), 5.39 (t, J = 5.4 Hz, 1H),

4.50 (bs, 1H), 4.21 (bs, 1H), 3.29 (q, J = 6.5 Hz, 2H), 2.84 (bs, 2H), 2.65 (t, J = 7.5 Hz,

2H), 2.20 (tt, J = 11.0, 3.5 Hz, 1H), 1.85 (p, J = 7.5 Hz, 2H), 1.72 (bs, 2H), 1.44 (bs, 2H).

TOF ES+ MS: (M + H) 515.1, (M + Na) 537.0. HPLC tR = 8.16 min, > 90%.

184

1-(1-(4-Chlorobenzyl)-1H-pyrrole-2-carbonyl)-N-((2,3-dihydro-1H-inden-2-

yl)methyl)azetidine-3-carboxamide (CCG-208793). Methyl 1-(1-(4-chlorobenzyl)-1H-

pyrrole-2-carbonyl)azetidine-3-carboxylate 29 (300 mg, 0.90 mmol) was dissolved in 200

proof EtOH (10 mL) and 10% aq. NaOH (10 mL) was added. The reaction was allowed

to stir at RT for 24 h, at which time the solvent was stripped off in vacuo and the residue

was taken up in H2O and acidified to pH 1 by addition of conc. HCl at 0°C. The resulting

precipitate was collected over a filter and dried by aspirator then high vacuum to afford

the desired carboxylic acid as a white powder. Yield: 250 mg, 0.78 mmol, 87%.

1-(1-(4-chlorobenzyl)-1H-pyrrole-2-carbonyl)azetidine-3-carboxylic acid (50 mg,

0.16 mmol) was added to DCM (5 mL), followed by indanyl amine 75 (29 mg, 0.17

mmol), TEA (66 µL, 0.47 mmol), EDCHCl (33 mg, 0.17 mmol), and HOBT (26 mg,

0.17 mmol). The reaction was allowed to stir at RT for 21 h, at which time the reaction

was concentrated and the residue taken up in EtOAc. This solution was then washed with

1M HCl (2X), H2O (3X) and 10% aq. Na2CO3 (3X), and brine (1X), dried (MgSO4), and

concentrated. The residue was purified by silica flash chromatography (4 g silica, 10% to

60% EtOAc:Hex) to provide the final compound as a white powder. Yield: 39 mg, 0.09

mmol, 56%. 1H NMR (500 MHz, Chloroform-d) δ 7.28 – 7.24 (m, 3H), 7.21 – 7.18 (m,

2H), 7.16 – 7.13 (m, 2H), 7.09 – 7.04 (m, 2H), 6.81 (dd, J = 2.6, 1.6 Hz, 1H), 6.52 (dd, J

= 4.0, 1.5 Hz, 1H), 6.19 – 6.16 (m, 1H), 5.56 (d, J = 9.0 Hz, 3H), 4.30 (bs, 4H), 3.42 (t, J

= 6.0 Hz, 2H), 3.22 (p, J = 7.4 Hz, 1H), 3.09 (q, J = 10.0, 9.3 Hz, 2H), 2.75 – 2.65 (m,

3H). TOF ES+ MS: (M + H) 448.2, (M + Na) 470.2. HPLC tR = 7.56 min, > 95%.

185

N1-(4-Chlorobenzyl)-N4-(2-(pyridin-4-yl)ethyl)piperidine-1,4-dicarboxamide

(208825). Ethyl 1-((4-chlorobenzyl)carbamoyl)piperidine-4-carboxylate 31a (810 mg,

2.50 mmol) was dissolved in 200 proof EtOH (20 mL) at RT, followed by the addition of

10% aq. NaOH, which elicited precipitation. The mixture was then stirred at 50 °C for 2

h over which time it became a solution. The reaction was then allowed to cool to RT and

the solvent was then removed in vacuo. The residue was taken up in H2O and acidified to

pH 1 with conc. HCl at 0 °C and the resulting precipitate was collected over a filter,

washed with cold 1 M aq. HCl, and dried over the filter then under high vacuum to give

the desired compound as a white powder. No further purification as necessary. Yield: 675

mg, 2.27 mmol, 91%.

1-((4-chlorobenzyl)carbamoyl)piperidine-4-carboxylic acid (60 mg, 0.20 mmol)

and TEA (93 µL, 0.61 mmol) were dissolved in DCM (5 mL), followed by EDCHCl (47

mg, 0.22 mmol), HOBT (37 mg, 0.22 mmol), and amine 27 (30 µL, 0.20 mmol). The

reaction was allowed to stir at RT for 15 h. After this time, the reaction was diluted with

EtOAc and washed with H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X), then dried

with MgSO4 and concentrated. The residue was then crystallized from EtOAc and

washed with Et2O to provide an off-white, slightly tanned solid. Yield: 42 mg, 0.11


(m, 4H), 7.11 (d, J = 4.4 Hz, 2H), 5.56 (s, 1H), 4.79 (d, J = 5.9 Hz, 1H), 4.37 (d, J = 5.2

Hz, 2H), 3.96 (d, J = 13.2 Hz, 2H), 3.55 (q, J = 6.4, 5.8 Hz, 2H), 2.85 – 2.78 (m, 4H),

2.19 (t, J = 11.5 Hz, 1H), 1.77 (d, J = 13.0 Hz, 2H), 1.63 (dd, J = 18.8, 5.5 Hz, 2H). TOF

ES+ MS: (M + H) 401.2, (M + Na) 423.2. HPLC tR = 4.61 min, > 95% purity.

186

N1-(4-Chlorobenzyl)-N

4-((2,3-dihydro-1H-inden-2-yl)methyl)piperidine-1,4-

dicarboxamide (CCG-208826). Ethyl 1-((4-chlorobenzyl)carbamoyl)piperidine-4-

carboxylate 31a (810 mg, 2.50 mmol) was dissolved in 200 proof EtOH (20 mL) at RT,

followed by the addition of 10% aq. NaOH, which elicited precipitation. The mixture was

then stirred at 50 °C for 2 h over which time it became a solution. The reaction was then

allowed to cool to RT and the solvent was then removed in vacuo. The residue was taken

up in H2O and acidified to pH 1 with conc. HCl at 0 °C and the resulting precipitate was

collected over a filter, washed with cold 1 M aq. HCl, and dried over the filter then under

high vacuum to give the desired compound as a white powder. No further purification as

necessary. Yield: 675 mg, 2.27 mmol, 91%.

1-((4-chlorobenzyl)carbamoyl)piperidine-4-carboxylic acid (60 mg, 0.20 mmol)

and TEA (93 µL, 0.61 mmol) were dissolved in DCM (5 mL), followed by EDCHCl (47


reaction was allowed to stir at RT for 16 h. After this time, the reaction was diluted with


with MgSO4 and concentrated. The residue was then crystallized from EtOAc and

washed with Et2O to provide a tan solid. Yield: 47 mg, 0.11 mmol, 54%. 1H NMR (500

MHz, Chloroform-d) δ 7.32 – 7.28 (m, 2H), 7.25 (d, J = 8.4 Hz, 2H), 7.21 – 7.18 (m,

2H), 7.16 – 7.13 (m, 2H), 5.57 (s, 1H), 4.39 (s, 2H), 3.98 (dt, J = 13.4, 3.7 Hz, 2H), 3.38

(t, J = 6.0 Hz, 2H), 3.08 (q, J = 10.3, 9.4 Hz, 2H), 2.85 (ddd, J = 13.3, 11.8, 2.8 Hz, 2H),

2.70 – 2.64 (m, 3H), 2.23 (ddt, J = 11.4, 7.7, 3.8 Hz, 1H), 1.83 – 1.58 (m, 4H). TOF ES+

MS: (M + H) 426.2. HPLC tR = 6.69 min, > 95%.

187

N1-(4-Chlorophenethyl)-N

4-(2-(pyridin-4-yl)ethyl)piperidine-1,4-dicarboxamide

(CCG-208827). Ethyl 1-((4-chlorophenethyl)carbamoyl)-piperidine-4-carboxylate 31b

(807 mg, 2.382 mmol) was dissolved in EtOH (20 mL), and 10% aq NaOH (15 mL) was

added. The resulting mixture was warmed to 50 °C and stirred for 2 h. The now

homogeneous solution was then concentrated in vacuo, a small amount of water was

added back, and the solution chilled in an ice bath. The solution was then acidified with

concentrated HCl, and the resulting precipitate was collected over a filter, washed with 1

M HCl and ice-cold water, filter-dried, then further dried under high vacuum to afford 1-

((4-chlorophenethyl)carbamoyl)piperidine-4-carboxylic acid as a white powder. Yield:

631 mg (85%). 1H NMR (400 MHz, DMSO-d6) δ 12.21 (s, 1H), 7.30 (d, J = 8.4 Hz, 2H),

7.18 (d, J = 8.4 Hz, 2H), 6.54 (t, J = 5.4 Hz, 1H), 3.80 (d, J = 13.3 Hz, 2H), 3.18 (q, J =

7.0 Hz, 2H), 2.76−2.62 (m, 4H), 2.36 (ddt, J = 11.0, 7.6, 3.9 Hz, 1H), 1.75−1.64 (m, 2H),

1.40−1.25 (m, 2H).

The carboxylic acid (60 mg, 0.193 mmol), TEA (81 μL, 0.579 mmol), EDCHCl

(41 mg, 0.212 mmol), HOBT (33 mg, 0.212 mmol), and 4-(2-aminoethyl)pyridine 27 (25

μL, 0.212 mmol) were all added sequentially to DCM (5 mL) at RT. This solution was

allowed to stir for 16 h at RT, after which time the reaction was diluted with ethyl acetate

and washed with water (3×), 10% aq sodium carbonate (3×), and brine (1×), then dried

(magnesium sulfate) and concentrated in vacuo. The resulting residue was then

crystallized from ethyl acetate and washed with diethyl ether to provide the title

compound as a tan solid. Yield: 46 mg (57%). 1HNMR (400 MHz, CDCl3) δ 8.61−8.26

(m, 2H), 7.30−7.19 (m, 2H), 7.17−7.01 (m, 4H), 5.91−5.66 (m, 1H), 4.59 (dd, J = 13.4,

5.6 Hz, 1H), 3.86 (d, J = 13.2 Hz, 2H), 3.53 (q, J = 6.7 Hz, 2H), 3.42 (q, J = 6.8 Hz, 2H),

188

2.82 (t, J = 6.8 Hz, 2H), 2.79−2.61 (m, 4H), 2.17 (t, J = 9.6 Hz, 1H), 1.73 (d, J = 12.9 Hz,

2H), 1.57 (qd, J = 12.9, 12.5, 3.8 Hz, 2H). TOF ES+ MS: (M + H) 415.2, (M + Na)

437.2. HPLC tR = 4.70 min, > 95%.

N1-(4-Chlorophenethyl)-N4-((2,3-dihydro-1H-inden-2-yl)methyl)piperidine-1,4-

dicarboxamide (CCG-208828). Ethyl 1-((4-chlorophenethyl)carbamoyl)-piperidine-4-

carboxylate 31b (807 mg, 2.382 mmol) was dissolved in EtOH (20 mL), and 10% aq

NaOH (15 mL) was added. The resulting mixture was warmed to 50 °C and stirred for 2

h. The now homogeneous solution was then concentrated in vacuo, a small amount of

water was added back, and the solution chilled in an ice bath. The solution was then

acidified with concentrated HCl, and the resulting precipitate was collected over a filter,

washed with 1 M HCl and ice-cold water, filter-dried, then further dried under high

vacuum to afford 1-((4-chlorophenethyl)carbamoyl)piperidine-4-carboxylic acid as a

white powder. Yield: 631 mg (85%). 1H NMR (400 MHz, DMSO-d6) δ 12.21 (s, 1H),

7.30 (d, J = 8.4 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 6.54 (t, J = 5.4 Hz, 1H), 3.80 (d, J =

13.3 Hz, 2H), 3.18 (q, J = 7.0 Hz, 2H), 2.76−2.62 (m, 4H), 2.36 (ddt, J = 11.0, 7.6, 3.9

Hz, 1H), 1.75−1.64 (m, 2H), 1.40−1.25 (m, 2H).

The carboxylic acid (60 mg, 0.19 mmol), TEA (81 μL, 0.58 mmol), EDCHCl (41

mg, 0.21 mmol), HOBT (33 mg, 0.21 mmol), and indanyl amine 75 (39 mg, 0.21 mmol)

were all added sequentially to DCM (5 mL) at RT. This solution was allowed to stir for

15 h at RT, after which time the reaction was diluted with EtOAc and washed with 05M

HCl (3X), H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X), then dried (magnesium

sulfate) and concentrated in vacuo. The resulting residue was then crystallized from

189

EtOAc to give tan crystals. Yield: 55 mg, 0.12 mmol, 64%. 1H NMR (500 MHz,

Chloroform-d) δ 7.28 – 7.27 (m, 2H), 7.21 – 7.18 (m, 2H), 7.16 – 7.12 (m, 4H), 5.54 (s,

1H), 4.45 (s, 1H), 3.88 (d, J = 9.9 Hz, 1H), 3.46 (q, J = 6.7 Hz, 2H), 3.38 (t, J = 6.0 Hz,

2H), 3.08 (q, J = 7.3, 6.5 Hz, 2H), 2.83 – 2.75 (m, 3H), 2.72 – 2.65 (m, 3H), 2.21 (tt, J =

11.5, 3.9 Hz, 1H), 1.79 (dd, J = 13.0, 3.2 Hz, 2H), 1.62 (td, J = 12.7, 12.2, 4.4 Hz, 4H).


N-Benzyl-1-(1-(4-chlorobenzyl)-1H-pyrrole-2-carbonyl)azetidine-3-carboxamide

(CCG-208829). Methyl 1-(1-(4-chlorobenzyl)-1H-pyrrole-2-carbonyl)azetidine-3-

carboxylate 29 (300 mg, 0.90 mmol) was dissolved in 200 proof EtOH (10 mL) and 10%

aq. NaOH (10 mL) was added. The reaction was allowed to stir at RT for 24 h, at which

time the solvent was stripped off in vacuo and the residue was taken up in H2O and

acidified to pH 1 by addition of conc. HCl at 0°C. The resulting precipitate was collected

over a filter and dried by aspirator then high vacuum to afford the desired carboxylic acid

as a white powder. Yield: 250 mg, 0.78 mmol, 87%.


0.16 mmol) was added to DCM (5 mL), followed by benzylamine (19 µL, 0.17 mmol),

TEA (66 µL, 0.47 mmol), EDCHCl (33 mg, 0.17 mmol), and HOBT (26 mg, 0.17

mmol). The reaction was allowed to stir at RT for 15 h, at which time the reaction was

concentrated and the residue taken up in EtOAc. This solution was then washed with

0.5M HCl (2X), H2O (3X) and 10% aq. Na2CO3 (3X), and brine (1X), dried (MgSO4),

and concentrated. The residue was purified by silica flash chromatography (4 g silica,

30% to 60% EtOAc:Hex) to provide the final compound as a white powder. Yield: 29

190

mg, 0.07 mmol, 45%. 1H NMR (500 MHz, Chloroform-d) δ 7.38 – 7.23 (m, 7H), 7.06 (d,

J = 8.2 Hz, 2H), 6.80 (s, 1H), 6.53 (d, J = 3.5 Hz, 1H), 6.19 – 6.16 (m, 1H), 5.82 (s, 1H),

5.54 (s, 2H), 4.57 (s, 1H), 4.48 (d, J = 5.7 Hz, 2H), 4.32 (s, 2H), 3.28 (p, J = 7.4 Hz, 1H).


1-(3-(4-Chlorophenyl)propanoyl)-N-(2-(pyridin-4-yl)ethyl)piperidine-4-

carboxamide (CCG-208846). Ethyl 1-(3-(4-chlorophenyl)propanoyl)piperidine-4-

carboxylate 33b (850 mg, 2.62 mmol) was dissolved in EtOH (10 mL), followed by the

addition of 10% aq. NaOH (10 mL). This was stirred 11 h at RT, after which time a

majority of the solvent was stripped off in vacuo. The remaining concentrated aq.

solution was chilled to 0 °C and acidified to pH<1 with conc. HCl. The aq. solution was

decanted off of the resulting gummy residue. H2O was then added, sonicated, and

decanted. The residue was then dried under high vacuum, and the thick viscous residue

was sonicated in Et2O to afford the carboxylic acid as a white solid that was collected

over a filter. Yield: 730 mg, 2.47 mmol, 94%.

1-(3-(4-chlorophenyl)propanoyl)piperidine-4-carboxylic acid (50 mg, 0.17 mmol)

was added to DCM (2 mL) at RT followed by TEA (71 µL, 0.51 mmol), EDC HCl (36


reaction was allowed to stir at RT for 14 h, after which time the DCM was removed in

vacuo and the residue taken up in EtOAc, which was subsequently washed with 0.5 M aq.

HCl (1X), H2O (2X), 10% aq. Na2CO3 (2X), and brine (1X), then dried (MgSO4) and

decanted. The material was crystallized from the dropwise addition of EtOAc to boiling

hexanes, then cooling of the solution. Yield: 42 mg, 0.11 mmol, 62%. 1H NMR (500

191

MHz, Chloroform-d) δ 8.52 – 8.48 (m, 2H), 7.29 – 7.20 (m, 2H), 7.17 – 7.07 (m, 4H),

5.66 (s, 1H), 4.57 (d, J = 12.9 Hz, 1H), 3.81 (d, J = 13.2 Hz, 1H), 3.54 (q, J = 5.9 Hz,

2H), 2.96 – 2.89 (m, 3H), 2.82 (t, J = 6.1 Hz, 2H), 2.63 – 2.54 (m, 3H), 2.22 (dt, J = 11.2,

5.8 Hz, 1H), 1.79 – 1.69 (m, 2H), 1.60 – 1.46 (m, 2H). TOF ES+ MS: 400.2 (M + H),

422.2 (M + Na). HPLC tR = 4.89 min, > 95% purity.

1-(3-(4-Chlorophenyl)propanoyl)-N-((2,3-dihydro-1H-inden-2-

yl)methyl)piperidine-4-carboxamide (CCG-208847). Ethyl 1-(3-(4-

chlorophenyl)propanoyl)piperidine-4-carboxylate 33b (850 mg, 2.62 mmol) was

dissolved in EtOH (10 mL), followed by the addition of 10% aq. NaOH (10 mL). This

was stirred 11 h at RT, after which time a majority of the solvent was stripped off in

vacuo. The remaining concentrated aq. solution was chilled to 0 °C and acidified to pH<1

with conc. HCl. The aq. solution was decanted off of the resulting gummy residue. H2O

was then added, sonicated, and decanted. The residue was then dried under high vacuum,

and the thick viscous residue was sonicated in Et2O to afford the carboxylic acid as a

white solid that was collected over a filter. Yield: 730 mg, 2.47 mmol, 94%.

The following were added sequentially to DCM (2 mL) at RT: 1-(2-(4-

chlorophenyl)acetyl)piperidine-4-carboxylic acid (50 mg, 0.17 mmol), TEA (71 µL, 0.51

mmol), EDCHCl (36 mg, 0.19 mmol), HOBT (29 mg, 0.19 mmol), and indanyl amine 75

(34 mg, 0.19 mmol). This solution was stirred a RT for 15 h, at which time the DCM was

removed in vacuo and the residue was diluted with EtOAc and washed with 1M HCl


concentrated. The crude residue was purified by crystallization from boiling EtOAc to

192

give crystals. Yield: 44 mg, 0.10 mmol, 61%. 1H NMR (500 MHz, Chloroform-d) δ 7.26

(d, J = 8.7 Hz, 2H), 7.21 – 7.18 (m, 2H), 7.17 – 7.12 (m, 4H), 5.54 (t, J = 5.9 Hz, 1H),

4.58 (dd, J = 13.6, 3.6 Hz, 1H), 3.83 (dt, J = 14.4, 3.9 Hz, 1H), 3.38 (t, J = 5.7 Hz, 2H),

3.08 (q, J = 7.5, 6.6 Hz, 2H), 3.00 – 2.89 (m, 3H), 2.74 – 2.65 (m, 3H), 2.60 (dd, J = 8.6,

6.3 Hz, 2H), 2.26 (tt, J = 11.4, 3.9 Hz, 1H), 1.83 – 1.74 (m, 2H), 1.60 – 1.47 (m, 2H).


1-(2-(4-Chlorophenyl)acetyl)-N-(2-(pyridin-4-yl)ethyl)piperidine-4-carboxamide

(CCG-208848). Ethyl 1-(2-(4-chlorophenyl)acetyl)piperidine-4-carboxylate 3a (811 mg,

2.62 mmol) was added to EtOH (15 mL) and 10% aq. NaOH (5 mL). This mixture was

heated to 50 °C and stirred for 5 h, then allowed to cool to RT. Solvent was removed in

vacuo until precipitation began to occur, at which point the material was cooled to 0 °C

and acidified to pH<1 by the dropwise addition of conc. HCl. The solution was decanted

off the resulting gum, and the residue was air dried, then dried under high vacuum to

afford a highly gummy material that could be solidified by sonication in Et2O to provide

the carboxylic acid as a white powder without further purification. Yield: 723 mg, 2.57

mmol, 98%.



mmol), EDCHCl (47 mg, 0.25 mmol), HOBT (37 mg, 0.25 mmol), and amine 27 (29

µL, 0.25 mmol). This solution was stirred a RT for 18 h, at which time the reaction was

diluted with EtOAc and washed with H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X),

then dried (MgSO4) and concentrated. The crude residue was purified by crystallization

193

from the dropwise addition of EtOAc to boiling hexanes, then cooling of the solution, to

give crystals. Yield: 40 mg, 0.105 mmol, 54%. 1H NMR (500 MHz, Chloroform-d) δ

8.52 (d, J = 4.7 Hz, 2H), 7.29 (d, J = 7.8 Hz, 2H), 7.17 (d, J = 8.1 Hz, 2H), 7.10 (d, J =

4.8 Hz, 2H), 5.50 (bs, 1H), 4.57 (d, J = 13.3 Hz, 1H), 3.86 (d, J = 13.5 Hz, 1H), 3.68 (s,

2H), 3.54 (q, J = 6.6 Hz, 2H), 3.03 – 2.96 (m, 1H), 2.82 (t, J = 6.9 Hz, 2H), 2.70 – 2.62

(m, 1H), 2.21 (tt, J = 11.1, 3.6 Hz, 1H), 1.77 (d, J = 12.3 Hz, 2H), 1.57 (qd, J = 12.2, 4.1

Hz, 1H), 1.46 (qd, J = 12.3, 3.9 Hz, 1H). TOF ES+ MS: 386.2 (M + H), 408.2 (M + Na).

HPLC tR = 6.87 min, > 95% purity.

1-(2-(4-Chlorophenyl)acetyl)-N-((2,3-dihydro-1H-inden-2-yl)methyl)piperidine-

4-carboxamide (CCG-208849). Ethyl 1-(2-(4-chlorophenyl)acetyl)piperidine-4-

carboxylate (811 mg, 2.62 mmol) was added to EtOH (15 mL) and 10% aq. NaOH (5

mL). This mixture was heated to 50 °C and stirred for 5 h, then allowed to cool to RT.

Solvent was removed in vacuo until precipitation began to occur, at which point the

material was cooled to 0 °C and acidified to pH<1 by the dropwise addition of conc. HCl.

The solution was decanted off the resulting gum, and the residue was air dried, then dried

under high vacuum to afford a highly gummy material that could be solidified by

sonication in Et2O to provide the carboxylic acid as a white powder without further

purification. Yield: 723 mg, 2.57 mmol, 98%.



mmol), EDCHCl (37 mg, 0.20 mmol), HOBT (30 mg, 0.20 mmol), and indanyl amine 75

(36 mg, 0.20 mmol). This solution was stirred a RT for 10 h, at which time the DCM was

194

removed in vacuo and the residue was diluted with EtOAc and washed with 0.5M HCl


concentrated. The crude residue was purified by crystallization from boiling EtOAc to

give crystals. Yield: 43 mg, 0.11 mmol, 59%. 1H NMR (500 MHz, Chloroform-d) δ 7.32

– 7.24 (m, 2H), 7.23 – 7.02 (m, 6H), 5.52 (t, J = 6.0 Hz, 1H), 4.63 – 4.53 (m, 1H), 3.95 –

3.82 (m, 1H), 3.69 (s, 2H), 3.37 (td, J = 6.0, 1.8 Hz, 2H), 3.13 – 2.97 (m, 3H), 2.70 – 2.57

(m, 4H), 2.24 (tt, J = 11.2, 3.9 Hz, 1H), 1.83 – 1.70 (m, 2H), 1.61 – 1.45 (m, 2H). TOF

ES+ MS: (M + H) 411.2. HPLC tR = 6.87 min, > 95%.

1-(1-(4-Chlorobenzyl)-1H-imidazole-2-carbonyl)-N-((2,3-dihydro-1H-inden-2-

yl)methyl)piperidine-4-carboxamide (CCG-208913). Ethyl 1-(4-chlorobenzyl)-1H-

imidazole-2-carboxylate 23b (7.28 g, 27.5 mmol), was dissolved in EtOH (10 mL) and

10% aq. NaOH (20 mL) and stirred at rt for 15 h. The solvent was then stripped off,

water was added, and the solution acidified with HCl. The resulting precipitate was

collected over a filter and washed with 1M HCl and dried to afford the carboxylic acid as

a white powder which was taken directly into the next step. Yield: 6.506 g, 27.5 mmol,

100%.

1-(4-chlorobenzyl)-1H-imidazole-2-carboxylic acid (50 mg, 0.21 mmol) was

added to DCM (5 mL), followed by N-((2,3-dihydro-1H-inden-2-yl)methyl)piperidine-4-

carboxamide (69 mg, 0.23 mmol), TEA (88 µL, 0.63 mmol), EDCHCl (45 mg, 0.23

mmol), and HOBT (36 mg, 0.23 mmol). The reaction was allowed to stir at RT for 20 h,

at which time the reaction was concentrated and the residue taken up in EtOAc. This

solution was then washed with H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X), dried

195

(MgSO4), and concentrated. The residue was purified by silica flash chromatography (10

g silica, 60 to 100% EtOAc:Hex) to provide the final compound as a white powder.


Hz, 2H), 7.21 – 7.12 (m, 6H), 7.07 (s, 1H), 6.96 (s, 1H), 5.67 – 5.61 (m, 1H), 5.38 (d, J =

5.6 Hz, 2H), 4.62 (t, J = 14.0 Hz, 2H), 3.37 (t, J = 5.8 Hz, 2H), 3.15 (t, J = 12.1 Hz, 1H),

3.07 (q, J = 9.6, 8.8 Hz, 2H), 2.87 – 2.79 (m, 1H), 2.71 – 2.65 (m, 3H), 2.34 (tt, J = 11.4,

4.0 Hz, 1H), 1.89 (d, J = 13.4 Hz, 1H), 1.82 – 1.75 (m, 1H), 1.61 (dtd, J = 36.5, 12.2, 3.5

Hz, 2H). TOF ES+ MS: (M + H) 477.2, (M + Na) 499.2. HPLC tR = 6.07 min, > 95%.

1-(1-(4-Chlorobenzyl)-1H-pyrrole-2-carbonyl)-N-((2,3-dihydro-1H-inden-2-

yl)methyl)piperidine-4-carboxamide (CCG-208914). 1-(1-(4-chlorobenzyl)-1H-pyrrole-

2-carbonyl)piperidine-4-carboxylic acid 25a (50 mg, 0.14 mmol) was added to DCM (5

mL), followed by indanyl amine 75 (29 mg, 0.16 mmol), TEA (60 µL, 0.43 mmol),


allowed to stir at RT for 20 h, at which time the reaction was concentrated and the residue

taken up in EtOAc. This solution was then washed with 1M HCl (3X), H2O (3X) and

10% aq. Na2CO3 (3X), and brine (1X), dried (MgSO4), and concentrated. The residue

was purified by silica flash chromatography (10 g silica, 60% EtOAc:Hex) to provide the

final compound as a white powder. Yield: 42 mg, 0.09 mmol, 61%. 1H NMR (500 MHz,

Chloroform-d) δ 7.29 – 7.25 (m, 2H), 7.23 – 7.17 (m, 2H), 7.17 – 7.13 (m, 2H), 7.05 (d, J

= 8.4 Hz, 2H), 6.81 – 6.80 (m, 1H), 6.34 (dd, J = 3.7, 1.6 Hz, 1H), 6.14 (d, J = 6.4 Hz,

1H), 5.52 (t, J = 5.0 Hz, 1H), 5.29 (s, 2H), 4.36 (d, J = 11.7 Hz, 2H), 3.38 (t, J = 6.0 Hz,

2H), 3.08 (q, J = 9.4, 8.9 Hz, 2H), 2.84 (t, J = 11.9 Hz, 2H), 2.73 – 2.65 (m, 3H), 2.25 (tq,

196

J = 11.2, 3.8 Hz, 1H), 1.77 – 1.68 (m, 2H), 1.43 (d, J = 11.7 Hz, 2H). TOF ES+ MS: (M

+ H) 476.2, (M + Na) 498.2. HPLC tR = 7.60 min, > 95%.

1-(1-(4-Chlorobenzyl)-1H-pyrrole-2-carbonyl)-N-(2-(pyridin-4-yl)ethyl)azetidine-

3-carboxamide (CCG-208915). Methyl 1-(1-(4-chlorobenzyl)-1H-pyrrole-2-

carbonyl)azetidine-3-carboxylate 29 (300 mg, 0.90 mmol) was dissolved in 200 proof

EtOH (10 mL) and 10% aq. NaOH (10 mL) was added. The reaction was allowed to stir

at RT for 24 h, at which time the solvent was stripped off in vacuo and the residue was

taken up in H2O and acidified to pH 1 by addition of conc. HCl at 0°C. The resulting

precipitate was collected over a filter and dried by aspirator then high vacuum to afford

the desired carboxylic acid as a white powder. Yield: 250 mg, 0.78 mmol, 87%.


0.16 mmol) was added to DCM (5 mL), followed by 4-(2-aminoethyl)pyridine (20 µL,

0.17 mmol), TEA (66 µL, 0.47 mmol), EDCHCl (33 mg, 0.17 mmol), and HOBT (26

mg, 0.17 mmol). The reaction was allowed to stir at RT for 17 h, at which time the

reaction was concentrated and the residue taken up in EtOAc. This solution was then

washed with H2O (3X) and 10% aq. Na2CO3 (3X), and brine (1X), dried (MgSO4), and

concentrated. The residue was purified by silica flash chromatography (4 g silica, 1% to

30% 7 M methanolic ammonia/EtOAc) to provide the final compound as a white powder.

Yield: 32 mg, 0.076 mmol, 48%. 1H NMR (500 MHz, Chloroform-d) δ 8.48 (s, 2H), 7.25

(d, J = 8.3 Hz, 2H), 7.10 (d, J = 4.1 Hz, 2H), 7.04 (d, J = 8.2 Hz, 2H), 6.80 (s, 1H), 6.50

(d, J = 3.9 Hz, 1H), 6.17 (t, J = 3.2 Hz, 1H), 5.97 (t, J = 5.2 Hz, 1H), 5.52 (s, 2H), 4.25

197

(bs, 4H), 3.58 (q, J = 6.6 Hz, 2H), 3.20 (p, J = 7.4 Hz, 1H), 2.84 (t, J = 6.9 Hz, 2H). TOF

ES+ MS: (M + H) 423.1, (M + Na) 445.1. HPLC rt = 5.09 min, >95% purity.

1-(1-(4-Chlorobenzyl)-1H-imidazole-2-carbonyl)-N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (CCG-208916). Ethyl 1-(4-chlorobenzyl)-1H-

imidazole-2-carboxylate 23b (7.28 g, 27.5 mmol), was dissolved in EtOH (10 mL) and

10% aq. NaOH (20 mL) and stirred at rt for 15 h. The solvent was then stripped off,

water was added, and the solution acidified with HCl. The resulting precipitate was

collected over a filter and washed with 1M HCl and dried to afford the carboxylic acid as

a white powder which was taken directly into the next step. Yield: 6.506 g, 27.5 mmol,

100%.

The following were added sequentially to DCM (5 mL): 1-(4-chlorobenzyl)-1H-

imidazole-2-carboxylic acid (73 mg, 0.309 mmol), N-(2-(pyridin-4-yl)ethyl)piperidine-4-

carboxamide dihydrochloride (100 mg, 0.371 mmol), DIPEA (162 μL, 0.927 mmol),

EDCHCl (65 mg, 0.340 mmol), and HOBt (52 mg, 0.340 mmol). The solution was

allowed to stir at RT for 24 h, after which time the DCM was stripped off in vacuo and

the resulting residue was taken up on 10% aq. Na2CO3. Material was then extracted with

EtOAc. The extract was dried (MgSO4) and concentrated in vacuo. The residue was then

triturated with EtOAc:Hex and collected over a filter. The impure yellow powder was

then recrystallized from EtOAc to provide the title compound as off-white crystals.

Yield: 89 mg (64%). 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 5.2 Hz, 2H), 7.28 (d, J =

8.3 Hz, 2H), 7.15–7.07 (m, 4H), 7.05 (s, 1H), 6.95 (s, 1H), 5.59 (t, J = 5.7 Hz, 1H), 5.35

(d,J = 8.9 Hz, 2H), 4.58 (d, J = 13.4 Hz, 2H), 3.53 (q, J = 6.7 Hz, 2H), 3.10 (t, J = 13.7

198

Hz, 1H), 2.87–2.71 (m, 3H), 2.29 (tt, J = 11.0, 3.4 Hz, 1H), 2.00–1.41 (m, 4H). TOF ES+

MS: (M + H) 452.2, (M + Na) 474.2. HPLC rt = 4.24 min, >90% purity.

1-(1-(4-Chlorobenzyl)-4-fluoro-1H-pyrrole-2-carbonyl)-N-((2,3-dihydro-1H-

inden-2-yl)methyl)piperidine-4-carboxamide (CCG-209020). The following reagents

were dissolved in DCM (3 mL) at RT: 1-(4-chlorobenzyl)-4-fluoro-1H-pyrrole-2-

carboxylic acid 136 (50 mg, 0.197 mmol), N-((2,3-dihydro-1H-inden-2-

yl)methyl)piperidine-4-carboxamide dihydrochloride (70 mg, 0.237 mmol), TEA (110

µL, 0.788 mmol), EDCHCl (45 mg, 0.237 mmol), and HOBT (36 mg, 0.237 mmol). The

reaction was allowed to stir for 16 h, after which time it was diluted with EtOAc and

washed with HCl (3X), H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X), then dried

(MgSO4) and concentrated. The residue was purified by silica flash chromatography (10

g silica, 5% to 50% EtOAc:Hex) to give the desired product as a white solid. Yield: 1H

NMR (400 MHz, Chloroform-d) δ 7.26 (d, J = 8.0 Hz, 2H), 7.21 – 7.09 (m, 4H), 7.05 (d,

J = 8.2 Hz, 2H), 6.56 – 6.50 (m, 1H), 6.05 (s, 1H), 5.54 (t, J = 5.1 Hz, 1H), 5.15 (s, 2H),

4.28 (d, J = 13.2 Hz, 2H), 3.36 (t, J = 5.7 Hz, 2H), 3.05 (q, J = 9.2 Hz, 2H), 2.82 (t, J =

11.7 Hz, 2H), 2.71 – 2.61 (m, 3H), 2.22 (tt, J = 11.1, 3.7 Hz, 1H), 1.71 (d, J = 12.0 Hz,

2H), 1.42 (d, J = 11.4 Hz, 2H). TOF ES+ MS: (M + H) 494.2. HPLC rt = 7.75 min, >95%

purity.

1-(1-(4-Chlorobenzyl)-4-fluoro-1H-pyrrole-2-carbonyl)-N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (CCG-209021). The following were added

sequentially to DCM (3 mL): 1-(4-chlorobenzyl)-4-fluoro-1H-pyrrole-2-carboxylic

199

acid 139 (50 mg, 0.20 mmol), TEA (0.11 mL, 0.79 mmol), EDCHCl (45 mg, 0.24

mmol), HOBT (36 mg, 0.24 mmol), and then amine dihydrochloride 36 (64 mg, 0.24

mmol). The reaction was allowed to stir at RT for 14 h, at which time it was diluted with

EtOAc, washed with H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X), dried with

MgSO4, and concentrated in vacuo. The residue was the purified by silica flash

chromatography (10 g silica, 1% 7 M methanolic ammonia/EtOAc) to provide the desired

material as a white powder. Yield: 48 mg (52%). 1H NMR (400 MHz, CDCl3) δ 8.54

(d, J = 4.3 Hz, 2H), 7.25–7.20 (m, 2H), 7.14 (d, J = 4.4 Hz, 2H), 7.06 (d, J = 6.9 Hz, 2H),

6.58–6.54 (m, 1H), 6.05 (s, 1H), 5.46–5.38 (m, 1H), 5.17 (s, 2H), 4.29 (d, J = 6.7 Hz,

2H), 3.55 (q, J = 7.3 Hz, 2H), 2.83 (dt, J = 17.9, 9.0 Hz, 4H), 2.27–2.15 (m, 1H), 1.77–

1.63 (m, 2H), 1.38 (d, J = 13.4 Hz, 2H). TOF ES+ MS: (M + H) 469.2, (M + Na) 491.2.

HPLC rt = 5.18 min, >95% purity.

1-(1-(4-Chlorobenzyl)-1H-pyrrolo[2,3-c]pyridine-2-carbonyl)-N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (CCG-211751). Azaindole ethyl ester 144 (300 mg,

0.953 mmol) was dissolved into ethanol (8 mL) at RT, followed by the addition of 10%

aqueous NaOH (8 mL), which initially caused precipitation, but homogeneity was

achieved over time. The reaction was allowed to stir at RT for 12 h, at which time the

solvent was removed in vacuo. The residue was taken up in a small amount of water (5

mL), the pH was adjusted to ∼4, and material was extracted with ethyl acetate (8×). The

extracts were combined and the solvent removed again in vacuo to provide the title

compound as a fine white powder. Yield: 226 mg (83%). 1H NMR (400 MHz, DMSO-

200

d6) δ 9.64 (s, 1H), 8.40 (d, J = 6.3 Hz, 1H), 8.26 (d, J = 6.3 Hz, 1H), 7.61 (s, 1H), 7.35 (d,

J = 8.5 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 6.06 (s, 2H).

The azaindole carboxylic acid (50 mg, 0.174 mmol) was dissolved into DCM (2

mL) at RT, followed by amine dihydrochloride 36 (59 mg, 0.192 mmol) and TEA (122

μL, 0.872 mmol). Once all material was in solution, EDCHCl (37 mg, 0.192 mmol) and

HOBT (29 mg, 0.192 mmol) were added and the reaction was allowed to stir for 18 h at

RT. At this time, the reaction was diluted with a solution of ethyl acetate/diethyl ether

(1:1) and washed with water (3×), 10% aq sodium carbonate (3×), and brine (1×), then

dried (magnesium sulfate) and concentrated in vacuo. The resulting residue was then

purified by flash chromatography (50 g silica, 5% 7.0 M methanolic ammonia/95% ethyl

acetate) to give the title compound as white solid. Yield: 53 mg (61%). 1H NMR (400

MHz, CDCl3) δ 8.78 (s, 1H), 8.49 (d, J = 5.4 Hz, 2H), 8.27 (d, J = 5.5 Hz, 1H), 7.51 (d, J

= 5.5 Hz, 1H), 7.21 (d, J = 8.2 Hz, 2H), 7.09 (d, J = 5.3 Hz, 2H), 7.05 (d, J = 8.2 Hz, 2H),

6.56 (s, 1H), 5.67 (t, J = 5.6 Hz, 1H), 5.48 (s, 2H), 4.57 (s, 1H), 3.91 (s, 1H), 3.53 (q, J =

6.6 Hz, 2H), 2.96−2.69 (m, 4H), 2.21 (tt, J =11.1, 7.5, 3.7 Hz, 1H), 1.88−1.69 (m, 1H),

1.67−1.46 (m, 2H), 1.33−1.22 (m, 1H). TOF ES+ MS: (M + H) 502.2. HPLC tR = 4.01

min, > 95%.

(R)-1-(1-(4-Chlorobenzoyl)pyrrolidine-2-carbonyl)-N-((2,3-dihydro-1H-inden-2-

yl)methyl)piperidine-4-carboxamide (CCG-211753). (R)-Ethyl 1-(1-(4-

chlorobenzoyl)pyrrolidine-2-carbonyl)piperidine-4-carboxylate (R)-40 (300 mg, 0.76

mmol) was dissolved in a 9:1 DCM:MeOH solution (20 mL), and a solution of

methanolic NaOH (92 mg NaOH/2mL MeOH) was added. This was stirred at RT for 2 h,

201

after which time the solvent was stripped off in vacuo to give a white solid that was

dissolved in a small amount of H2O and acidified by the addition of conc. HCl, and the

material was extracted with EtOAc (3X). The combined organic extracts were dried

(MgSO4) and concentrated. The clear oily residue was then dissolved in minimum EtOAc

and precipitated with Hex to afford a white powder. No further purification necessary.

Yield: 260 mg, 0.71 mmol, 93%.

(R)-1-(4-chlorobenzoyl)pyrrolidine-2-carboxylic acid (50 mg, 0.14 mmol) was

added to DCM (5 mL), followed by the sequential addition of TEA (76 µL, 0.55 mmol),

HATU (63 mg, 0.16 mmol), and indanyl amine 75 (24 mg, 0.16 mmol). The reaction

was stirred for 22 h at RT, after which time the reaction was diluted in sat. aq. NH4Cl and

extracted with EtOAc. The organic extract was then washed with 1M HCl (3X), H2O

(3X), 10% aq. Na2CO3 (3X), and sat. aq. Na2CO3 (1X), then dried with MgSO4 and

concentrated. The residue was purified via silica flash chromatography (4 g silica, 5% to

20% 7 M methanolic ammonia:EtOAc) to give the desired compound as a white powder.


Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.18 – 7.11 (m, 4H), 6.04 (s, 0.5H), 5.75 – 5.67 (m,

0.5H), 5.03 (m, 1H), 4.60 (d, J = 13.3 Hz, 0.5H), 4.47 (d, J = 13.6 Hz, 0.5H), 4.07 (dd, J

= 30.7, 13.8 Hz, 1H), 3.80 (m, 0.5H), 3.68 (m, 1.5H), 3.51 (m, 1H), 3.32 (m, 2H), 3.04

(m, 2H), 2.85 – 2.74 (m, 1H), 2.72 – 2.56 (m, 3H), 2.43 (m, 0.5H), 2.36 – 2.14 (m, 1.5H),

2.14 – 2.00 (m, 2H), 2.02 – 1.76 (m, 5H), 1.65 (dd, J = 29.2, 12.8 Hz, 2H). TOF ES+ MS:

(M + H) 494.2. HPLC tR = 6.37 min, > 95% purity.

202

(S)-1-(1-(4-Chlorobenzoyl)pyrrolidine-2-carbonyl)-N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (CCG-211751). (S)-Ethyl 1-(1-(4-

chlorobenzoyl)pyrrolidine-2-carbonyl)piperidine-4-carboxylate (S)-40 (300 mg, 0.76







ad precipitated with Hex to afford a white powder. No further purification necessary.

Yield: 265 mg, 0.73 mmol, 95%.

(S)-1-(4-chlorobenzoyl)pyrrolidine-2-carboxylic acid (50 mg, 0.14 mmol) was


HATU (63 mg, 0.16 mmol), and amine 27 (20 µL, 0.16 mmol). The reaction was stirred

for 17 h at RT, after which time the reaction was diluted in sat. aq. NH4Cl and extracted

with EtOAc. The organic extract was then washed with 10% aq. NH4Cl (2X), 10% aq.

Na2CO3 (3X), and sat. aq. Na2CO3 (1X), then dried with MgSO4 and concentrated. The

residue was purified via silica flash chromatography (4 g silica, 5% to 20% 7 M

methanolic ammonia:EtOAc) to give the desired compound as a white powder. Yield: 42

mg, 0.09 mmol, 65%. 1H NMR (500 MHz, Chloroform-d) δ 8.52 (m, 2H), 7.53 (d, J =

8.0 Hz, 2H), 7.37 (t, J = 7.0 Hz, 2H), 7.16 – 7.09 (m, 2H), 5.91 (d, J = 2.1 Hz, 0.5H),

5.58 – 5.49 (m, 0.5H), 5.07 – 4.97 (m, 1H), 4.63 (d, J = 14.5 Hz, 0.5H), 4.48 (d, J = 13.9

Hz, 1H), 4.09 (dd, J = 41.8, 14.7 Hz, 2H), 3.68 (t, J = 9.9 Hz, 1H), 3.54 (m, 3H), 3.32 –

203

3.21 (m, 0.5H), 3.12 (t, J = 12.8 Hz, 0.5H), 2.83 (dt, J = 14.6, 6.8 Hz, 3H), 2.64 (t, J =

12.6 Hz, 0.5H), 2.47 –1.15 (m, 7H). TOF ES+ MS: (M + H) 469.3, (M + Na) 491.2.


(S)-1-(1-(4-Chlorobenzoyl)pyrrolidine-2-carbonyl)-N-((2,3-dihydro-1H-inden-2-

yl)methyl)piperidine-4-carboxamide (CCG-211755). (S)-Ethyl 1-(1-(4-

chlorobenzoyl)pyrrolidine-2-carbonyl)piperidine-4-carboxylate (S)-40 (300 mg, 0.76







ad precipitated with Hex to afford a white powder. No further purification necessary.

Yield: 265 mg, 0.73 mmol, 95%.

(S)-1-(4-chlorobenzoyl)pyrrolidine-2-carboxylic acid (50 mg, 0.14 mmol) was


HATU (63 mg, 0.16 mmol), and indanyl amine 75 (24 mg, 0.16 mmol). The reaction

was stirred for 19 h at RT, after which time the reaction was diluted in sat. aq. NH4Cl and

extracted with EtOAc. The organic extract was then washed with 1M HCl (3X), H2O

(3X), 10% aq. Na2CO3 (3X), and sat. aq. Na2CO3 (1X), then dried with MgSO4 and

concentrated. The residue was purified via silica flash chromatography (4 g silica, 5% to

20% 7 M methanolic ammonia:EtOAc) to give the desired compound as a white powder.

204

Yield: 44 mg, 0.09 mmol, 68%. 1H NMR (400 MHz, Chloroform-d) δ 7.61 – 7.54 (m,

1H), 7.54 – 7.49 (m, 1H), 7.44 – 7.37 (m, 1H), 7.37 – 7.28 (m, 2H), 7.25 – 7.06 (m, 4H),

6.14 – 6.02 (m, 0.5H), 5.81 – 5.70 (m, 0.5H), 5.19 – 4.96 (m, 1H), 4.60 (d, J = 13.6 Hz,

0.5H), 4.47 (d, J = 13.7 Hz, 0.5H), 4.06 (dd, J = 30.8, 14.3 Hz, 1H), 3.73 – 3.61 (m, 1H),

3.59 – 3.43 (m, 1H), 3.42 – 3.21 (m, 2H), 3.19 – 2.94 (m, 3H), 2.76 – 2.53 (m, 3H), 2.35

– 2.14 (m, 2H), 2.11 – 1.77 (m, 5H), 1.77 – 1.51 (m, 2H). TOF ES+ MS: (M + H) 494.2.


(R)-1-(1-(4-Chlorobenzoyl)pyrrolidine-2-carbonyl)-N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (CCG-211756). (R)-Ethyl 1-(1-(4-

chlorobenzoyl)pyrrolidine-2-carbonyl)piperidine-4-carboxylate (R)-40 (300 mg, 0.76







and precipitated with Hex to afford a white powder. No further purification necessary.

Yield: 260 mg, 0.71 mmol, 93%.

(R)-1-(4-chlorobenzoyl)pyrrolidine-2-carboxylic acid (50 mg, 0.14 mmol) was


HATU (63 mg, 0.16 mmol), and amine 27 (20 µL, 0.16 mmol). The reaction was stirred

for 18 h at RT, after which time the reaction was diluted in sat. aq. NH4Cl and extracted

205

with EtOAc. The organic extract was then washed with 10% aq. NH4Cl (2X), 10% aq.

Na2CO3 (3X), and sat. aq. Na2CO3 (1X), then dried with MgSO4 and concentrated. The

residue was purified via silica flash chromatography (4 g silica, 5% to 20% 7 M

methanolic ammonia:EtOAc) to give the desired compound as a white powder. Yield: 41

mg, 0.09 mmol, 62%. 1H NMR (500 MHz, Chloroform-d) δ 8.59 – 8.47 (m, 2H), 7.53

(d, J = 8.1 Hz, 2H), 7.38 (t, J = 7.6 Hz, 2H), 7.12 (dd, J = 9.5, 4.8 Hz, 2H), 5.90 (s, 0.5H),

5.50 (s, 0.5H), 5.10 – 4.99 (m, 1H), 4.63 (d, J = 13.3 Hz, 0.5H), 4.48 (d, J = 13.2 Hz,

1H), 4.09 (dd, J = 40.4, 14.2 Hz, 2H), 3.70 (t, J = 8.6 Hz, 1H), 3.54 (m, 3H), 3.29 (t, J =

12.8 Hz, 0.5H), 3.12 (t, J = 12.7 Hz, 0.5H), 2.83 (dt, J = 14.7, 6.6 Hz, 3H), 2.64 (t, J =

12.6 Hz, 0.5H), 2.40-1.20 (m, 7H). TOF ES+ MS: (M + H) 469.3, (M + Na) 491.3. HPLC

tR = 4.26 min, > 95% purity.

(S)-1-(1-(4-Chlorobenzyl)pyrrolidine-2-carbonyl)-N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (CCG-211757). (S)-1-(4-chlorobenzyl)pyrrolidine-2-

carboxylic acid (S)-38 (63 mg, 0.27 mmol) was added to DCM (2 mL), followed by the

sequential addition of TEA (150 µL, 1.06 mmol), HATU (111 mg, 0.29 mmol), and

amine dihydrochloride 36 (75 mg, 0.28 mmol). The reaction was stirred for 24 h at RT,

after which time the reaction was diluted in sat. aq. NH4Cl and extracted with EtOAc.

The organic extract was then washed with 10% aq. NH4Cl (2X), 10% aq. Na2CO3 (3X),

and sat. aq. Na2CO3 (1X), then dried with MgSO4 and concentrated. The residue was

purified via silica flash chromatography (4 g silica, 5% to 20% 7 M methanolic

ammonia/EtOAc) to give a slightly yellow oil that was triturated from Et2O/Hex to

provide a white powder. Yield: 64 mg, 0.14 mmol, 53%. 1H NMR (500 MHz,

206

Chloroform-d) δ 8.48 (d, J = 4.6 Hz, 2H), 7.31 – 7.22 (m, 4H), 7.11 (bs, 2H), 5.82 (bs,

1H), 4.52 (bs, 1H), 4.13 (d, J = 11.9 Hz, 1H), 3.89 (dd, J = 18.3, 13.1 Hz, 1H), 3.58 –

3.49 (m, 2H), 3.46 – 3.34 (m, 2H), 3.10 – 3.00 (m, 1H), 2.94 (d, J = 9.3 Hz, 1H), 2.87 –

2.79 (m, 2H), 2.63 – 2.52 (m, 1H), 2.33 – 2.26 (m, 2H), 2.18 – 2.08 (m, 1H), 1.89 – 1.75

(m, 4H), 1.60 – 1.50 (m, 2H). TOF ES+ MS: (M + H) 455.2, (M + Na) 477.2. HPLC tR =

3.39 min, > 90% purity.

(R)-1-(1-(4-Chlorobenzyl)pyrrolidine-2-carbonyl)-N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (CCG-211758). (R)-1-(4-chlorobenzyl)pyrrolidine-2-

carboxylic acid (R)-38 (64 mg, 0.27 mmol) was added to DCM (2 mL), followed by the

sequential addition of TEA (150 µL, 1.06 mmol), HATU (111 mg, 0.29 mmol), and

amine dihydrochloride 36 (75 mg, 0.28 mmol). The reaction was stirred for 20 h at RT,

after which time the reaction was diluted in sat. aq. NH4Cl and extracted with EtOAc.

The organic extract was then washed with 10% aq. NH4Cl (2X), 10% aq. Na2CO3 (3X),

and sat. aq. Na2CO3 (1X), then dried with MgSO4 and concentrated. The residue was

purified via silica flash chromatography (4 g silica, 5% to 20% 7 M methanolic

ammonia:EtOAc) to give a slightly yellow oil that was triturated from Et2O/Hex to

provide a white powder. Yield: 75 mg, 0.17 mmol, 62%. 1H NMR (500 MHz,

Chloroform-d) δ 8.52 (d, J = 5.1 Hz, 2H), 7.26 (s, 4H), 7.12 (s, 2H), 5.57 (s, 1H), 4.55 (d,

J = 11.7 Hz, 1H), 4.15 (bs, 1H), 3.90 (dd, J = 18.8, 13.1 Hz, 1H), 3.61 – 3.50 (m, 2H),

3.39 (d, J = 12.0 Hz, 2H), 3.06 (d, J = 7.0 Hz, 1H), 2.98 – 2.91 (m, 1H), 2.84 (q, J = 6.2

Hz, 2H), 2.59 (q, J = 13.9, 13.3 Hz, 1H), 2.33 – 2.21 (m, 2H), 2.14 (s, 1H), 1.80 – 1.77

207

(m, 2H), 1.59 – 1.48 (m, 2H). TOF ES+ MS: (M + H) 455.2, (M + Na) 477.2. HPLC tR =

3.44 min, > 90% purity.

(E)-(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-(4-(pyridin-4-yl)but-1-en-1-

yl)piperidin-1-yl)methanone (CCG-211822). (E)-tert-butyl 4-(4-(pyridin-4-yl)but-1-en-

1-yl)piperidine-1-carboxylate 99b (100 mg, 0.316 mmol) was dissolved in anhydrous

1,4-dioxane (1 mL) and 4 M HCl in 1,4-dioxane (0.55 mL, 2.21 mmol) was added. The

reaction was allowed to stir for 1 h, at which time the solvent was stripped off and

replaced with Et2O. The oily residue was solidified with sonication, washed (decantation)

with Et2O (3X) and then dried under high vacuum to give the dihydrochloride as a white

powder. Yield: 91 mg, 0.316 mmol, 100%.

(E)-4-(4-(pyridin-4-yl)but-1-en-1-yl)piperidine dihydrochloride (75 mg, 0.259

mmol) was dissolved in DCM (10 mL) and TEA (217 µL, 1.56 mmol), followed by the

indole carboxylic acid 4a (89 mg, 0.311 mmol), EDCHCl (60 mg, 0.311 mmol), and

HOBT (48 mg, 0.311 mmol), and the reaction was allowed to stir at RT for 18 h. After

this time, the reaction was diluted with EtOAc and washed with H2O (3X), 10% Na2CO3

(3X), and brine (1X), then dried (Na2SO4) and concentrated in vacuo. The resulting

residue was purified via silica flash chromatography (10 g silica, 1% to 20% methanolic

ammonia:EtOAc) to provide a clear, colorless oil. Yield: 72 mg, 0.148 mmol, 57%. 1H

NMR (400 MHz, Chloroform-d) δ 8.54 – 8.39 (m, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.35 (d,

J = 8.2 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.21 – 7.10 (m, 3H), 7.08 (s, 2H), 7.01 (d, J =

8.0 Hz, 2H), 6.59 (s, 1H), 5.46 (s, 2H), 5.37 – 5.31 (m, 1H), 5.26 (d, J = 11 Hz, 1H), 4.57

(bs, 1H), 4.04 (bs, 1H), 2.85 (bs, 2H), 2.65 (t, J = 7.3 Hz, 2H), 2.39 – 2.23 (m, 2H), 2.07

208

(s, 1H), 1.64 (bs, 1H), 1.41 (bs, 1H), 1.08 (bs, 1H), 0.64 (bs, 1H). TOF ES+ MS: (M + H)

484.2. HPLC tR = 6.24 min, > 95% purity.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(2-(pyridin-4-yl)ethyl)-1,2,3,6-

tetrahydropyridine-4-carboxamide (CCG-211823). Ethyl 1-(1-(4-chlorobenzyl)-1H-

indole-2-carbonyl)-1,2,3,6-tetrahydropyridine-4-carboxylate (53 mg, 0.125 mmol) was

dissolved in EtOH (5 mL), followed by the addition of 10% aq. NaOH (2 mL). This

reaction was allowed to stir for 4 h, at which time the solvent was removed in vacuo. The

residue was dissolved in H2O and acidified with conc. HCl at 0 °C. The resulting

precipitate was collected over a filter and dried under high vacuum to give a white


1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)-N-(2-(pyridin-4-yl)ethyl)-1,2,3,6-

tetrahydropyridine-4-carboxylic acid (33 mg, 0.084 mmol) was dissolved in DCM (5 mL)

and TEA (35 µL, 0.251 mmol), followed by EDCHCl (18 mg, 0.092 mmol), and HOBT

(14 mg, 0.092 mmol), and the reaction was allowed to stir at RT for 21 h. After this time,

the reaction was diluted with EtOAc:Et2O and washed with H2O (2X), 10% Na2CO3


residue was purified via silica flash chromatography (4 g silica, 80% to 100%

EtOAc:Hex) to provide a fine white powder. Yield: 38 mg, 0.076 mmol, 91%. 1H NMR

(400 MHz, Chloroform-d) δ 8.51 (d, J = 4.1 Hz, 2H), 7.66 (d, J = 7.9 Hz, 1H), 7.30 –

7.05 (m, 8H), 6.97 (d, J = 8.1 Hz, 2H), 6.78 (s, 1H), 5.77 (s, 1H), 5.51 (s, 2H), 4.83 (bs,

1H), 3.89 (bs, 1H), 3.61 – 3.43 (m, 3H), 2.99 (s, 1H), 2.84 (t, J = 6.9 Hz, 2H), 2.16 (bs,

209

1H), 1.94 (bs, 1H). TOF ES+ MS: (M + H) 499.2, (M + Na) 521.2. HPLC tR = 5.46 min,

> 95%.

1-(2-((4-Chlorophenyl)amino)benzoyl)-N-(2-(pyridin-4-yl)ethyl)piperidine-4-

carboxamide (CCG-211824). Ethyl 1-(2-((4-chlorophenyl)amino)benzoyl)piperidine-4-

carboxylate (72 mg, 0.19 mmol) was dissolved in EtOH (5 mL), followed by the addition

of 10% aq. NaOH (2 mL). This reaction was allowed to stir at RT for 24 h, after which

time the solvent was stripped off in vacuo and the residue was taken up in H2O and

acidified to pH<1 with conc. HCl at 0 °C. The resulting white precipitate was collected

over a filter, dried via aspirator then high vacuum to afford the carboxylic acid as a white

powder requiring no further purification. Yield: 47 mg, 0.13 mmol, 70%.

1-(2-((4-chlorophenyl)amino)benzoyl)piperidine-4-carboxylic acid (36 mg, 0.10

mmol), 4-(2-aminoethyl)pyridine (13 µL, 0.11 mmol), TEA (42 µL, 0.30 mmol),

EDCHCl (21 mg, 0.11 mmol), and HOBT (17 mg, 0.11 mmol) were all added to DCM

(5 mL) in the listed order and stirred at RT for 20 h, at which time the reaction was

diluted with 1:1 EtOAc:Et2O and washed with 0.5 M aq. HCl (1X), H2O (1X), 10% aq.

Na2CO3 (3X), and brine (1X). The organic phase was then dried (MgSO4) and


80% to 100% EtOAc:Hex gradient) to give the title compound as a clear, colorless oil.


Hz, 2H), 7.32 (d, J = 8.3 Hz, 1H), 7.26 – 7.19 (m, 3H), 7.17 (d, J = 7.6 Hz, 1H), 7.13 –

7.09 (m, 2H), 7.00 (d, J = 8.6 Hz, 2H), 6.89 (t, J = 7.4 Hz, 1H), 5.56 (s, 1H), 4.23 (bs,

2H), 3.53 (q, J = 6.6 Hz, 2H), 2.93 (s, 2H), 2.82 (t, J = 6.9 Hz, 2H), 2.28 (tt, J = 11.2, 3.7

210

Hz, 1H), 1.80 – 1.66 (m, 4H). TOF ES+ MS: (M + H) 463.2, (M + Na) 485.2. HPLC tR =

5.23 min, > 95%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-4-methyl-N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (CCG-211825). 1-(1-(4-chlorobenzyl)-1H-indole-2-

carbonyl)-4-methylpiperidine-4-carboxylic acid (100 mg, 0.243 mmol), 4-(2-

aminoethyl)pyridine (32 µL, 0.268 mmol), TEA (100 µL, 0.730 mmol), EDCHCl (51

mg, 0.268 mmol), and HOBT (41 mg, 0.268 mmol) were dissolved in DCM (5 mL) and

stirred at RT for 16 h. After this time, the reaction was diluted with a 2:1 solution of

EtOAc:Et2O and washed with H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X), then


chromatography (4 g silica, 1% to 5% methanolic ammonia:EtOAc) to give a clear

colorless oil. Yield: 113 mg, 0.219 mmol, 90%. 1H NMR (500 MHz, Chloroform-d) δ


7.7 Hz, 1H), 7.19 – 7.12 (m, 3H), 7.08 (d, J = 4.8 Hz, 2H), 7.00 (d, J = 8.0 Hz, 2H), 6.58

(s, 1H), 5.95 (t, J = 5.6 Hz, 1H), 5.44 (s, 2H), 4.02 (s, 1H), 3.68 (s, 1H), 3.52 (q, J = 6.5

Hz, 2H), 3.22 (s, 2H), 2.81 (t, J = 6.9 Hz, 2H), 2.22 (s, 1H), 1.86 (s, 1H), 1.67 (s, 1H),

1.28 (s, 1H), 1.05 (s, 3H), 0.77 (s, 1H). TOF ES+ MS: (M + H) 515.0. HPLC tR = 5.49

min, > 95%.

(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-(3-(pyridin-4-yl)propyl)piperazin-1-

yl)methanone (CCG-211826). t-Butyl 4-(3-(pyridin-4-yl)propyl)piperazine-1-carboxylate

(160 mg, 0.524 mmol) was dissolved in 1,4-dioxane (2 mL) at RT, and a 4 M HCl in 1,4-

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dioxane solution (1.31 mL, 5.24 mmol) was added. The reaction was allowed to stir for

15 min, over which time precipitation occurred. The solvent was removed in vacuo and

dried under high vacuum to give the trishydrochloride salt as a tan powder. Yield: 168

mg, 0.524 mmol, 100%.

1-(4-Chlorobenzyl)-1H-indole-2-carboxylic acid (91 mg, 0.318 mmol), 4-(3-

(pyridin-4-yl)propyl)piperazine trishydrochloride (100 mg, 0.318 mmol), TEA (266 µL,

1.907 mmol), EDCHCl (67 mg, 0.350 mmol), and HOBT (54 mg, 0.350 mmol) were

dissolved in DCM (5 mL) and stirred at RT for 14 h. After this time, the reaction was

diluted with a 2:1 solution of EtOAc:Et2O and washed with H2O (3X), 10% aq. Na2CO3

(3X), and brine (1X), then dried (MgSO4) and concentrated. The residue was purified by

silica flash chromatography (4 g silica, 1% to 5% MeOH:EtOAc) to give a clear colorless

oil. Yield: 94 mg, 0.199 mmol, 63%. 1H NMR (500 MHz, Chloroform-d) δ 8.49 (d, J =

5.2 Hz, 2H), 7.65 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.31 – 7.26 (m, 1H), 7.19

(dd, J = 13.2, 7.9 Hz, 3H), 7.11 (d, J = 5.2 Hz, 2H), 7.03 (d, J = 8.2 Hz, 2H), 6.62 (s, 1H),

5.49 (s, 2H), 3.64 (bs, 4H), 2.64 (t, J = 7.6 Hz, 2H), 2.36 – 2.26 (m, 4H), 2.00 (bs, 2H),

1.79 (p, J = 7.5 Hz, 2H). TOF ES+ MS: (M + H) 473.2, (M + Na) 495.2. HPLC tR = 4.78

min, > 95%.

(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-phenethylpiperazin-1-yl)methanone (CCG-

211827). 1-(4-Chlorobenzyl)-1H-indole-2-carboxylic acid (100 mg, 0.350 mmol), N-(2-

phenylethyl)piperazine (73 µL, 0.385 mmol), TEA (146 µL, 1.050 mmol), EDCHCl (74

mg, 0.385 mmol), and HOBT (59 mg, 0.385 mmol) were dissolved in DCM (6 mL) and

stirred at RT for 14 h. After this time, the reaction was diluted with a 2:1 solution of

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EtOAc:Et2O and washed with H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X), then


chromatography (4 g silica, 60% EtOAc:Hex) to give a clear colorless oil. Yield: 109 mg,

0.238 mmol, 68%. 1H NMR (500 MHz, Chloroform-d) δ 7.67 (d, J = 7.9 Hz, 1H), 7.39

(d, J = 8.4 Hz, 1H), 7.33 – 7.26 (m, 3H), 7.20 (dt, J = 19.0, 7.8 Hz, 6H), 7.05 (d, J = 8.3

Hz, 2H), 6.65 (s, 1H), 5.50 (s, 2H), 3.68 (s, 4H), 2.77 (dd, J = 9.6, 6.5 Hz, 2H), 2.57 (dd,

J = 9.7, 6.5 Hz, 2H), 2.28 (d, J = 147.4 Hz, 5H). TOF ES+ MS: (M + H) 458.2. HPLC tR

= 5.76 min, > 95%.

(Z)-(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-(4-(pyridin-4-yl)but-1-en-1-

yl)piperidin-1-yl)methanone (CCG-211829). (Z)-tert-butyl 4-(4-(pyridin-4-yl)but-1-en-1-

yl)piperidine-1-carboxylate 99a (100 mg, 0.316 mmol) was dissolved in anhydrous 1,4-

dioxane (1 mL) and 4 M HCl in 1,4-dioxane (0.55 mL, 2.21 mmol) was added. The

reaction was allowed to stir for 1 h, at which time the solvent was stripped off and

replaced with Et2O. The oily residue was solidified with sonication, washed (decantation)

with Et2O (3X) and then dried under high vacuum to give the dihydrochloride as a white


(Z)-4-(4-(pyridin-4-yl)but-1-en-1-yl)piperidine dihydrochloride (75 mg, 0.259

mmol) was dissolved in DCM (10 mL) and TEA (217 µL, 1.56 mmol), followed by the

indole carboxylic acid 4a (89 mg, 0.311 mmol), EDCHCl (60 mg, 0.311 mmol), and

HOBT (48 mg, 0.311 mmol), and the reaction was allowed to stir at RT for 16 h. After

this time, the reaction was diluted with EtOAc and washed with H2O (3X), 10% Na2CO3


213

residue was purified via silica flash chromatography (10 g silica, 1% to 20% methanolic

ammonia:EtOAc) to provide a clear, colorless oil. Yield: 54 mg, 0.111 mmol, 43%. 1H


(d, J = 8.3 Hz, 1H), 7.28 – 7.22 (m, 1H), 7.17 (dd, J = 15.8, 7.9 Hz, 3H), 7.09 (d, J = 5.5

Hz, 2H), 7.01 (d, J = 8.3 Hz, 2H), 6.58 (s, 1H), 5.46 (s, 2H), 5.32 (dt, J = 8.1, 7.4 Hz,

1H), 5.11 (t, J = 8.1 Hz, 1H), 4.54 (bs, 1H), 4.05 (bs, 1H), 2.78 (bs, 2H), 2.64 (t, J = 7.2

Hz, 2H), 2.36 (q, J = 7.4 Hz, 2H), 2.31 – 2.19 (m, 1H), 1.43 (bs, 1H), 1.12 (bs, 2H), 0.64

(bs, 1H). TOF ES+ MS: (M + H) 484.2. HPLC tR = 6.29 min, > 95%.

N-(1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)piperidin-4-yl)-3-(pyridin-4-

yl)propanamide (CCG-212052). t-Butyl 4-(3-(pyridin-4-yl)propanamido)piperidine-1-

carboxylate (250 mg, 0.750 mmol) was dissolved in 1,4-dioxane (1 mL) and 4 M HCl

(1.0 mL, 4.12 mmol) in 1,4-dioxane was added. The reaction was allowed to stir for 1 h,

then the solvent was removed and replaced with Et2O and the oily material was solidified

with sonication. Washing with Et2O and thorough drying under high vacuum afforded the

desired dihydrochloride salt. Yield: 230 mg, 0.750 mmol, 100%.

4-(3-(pyridin-4-yl)propanamido)piperidine dihydrochloride (100 mg, 0.327

mmol), 1-(4-chlorobenzyl)-1H-indole-2-carboxylic acid 4a (112 mg, 0.392 mmol), TEA

(273 µL, 1.959 mmol), EDCHCl (75 mg, 0.392 mmol), and HOBT (60 mg, 0.392 mmol)

were all dissolved in DCM (10 mL) and stirred at RT for 22 h, after which time the DCM

was removed in vacuo and the residue was taken up in EtOAc and washed with H2O

(2X), 10% Na2CO3 (3X), and brine (1X), then dried (MgSO4) and concentrated. The

resulting residue was purified via silica flash chromatography (10 g silica, 1% to 20%

214

methanolic ammonia:EtOAc) to provide a white solid. Yield: 100 mg, 0.20 mmol, 61%.


7.41 (d, J = 8.4 Hz, 1H), 7.30 (t, J = 7.7 Hz, 1H), 7.22 – 7.17 (m, 3H), 7.14 – 7.11 (m,

2H), 7.07 – 7.03 (m, 2H), 6.59 (d, J = 1.8 Hz, 1H), 5.48 (s, 2H), 5.30 (d, J = 8.0 Hz, 1H),

4.50 (s, 1H), 4.09 (s, 1H), 3.89 (d, J = 7.1 Hz, 1H), 2.95 (t, J = 7.3 Hz, 2H), 2.82 (s, 2H),

2.45 (t, J = 7.1 Hz, 2H), 1.83 (bs, 1H), 1.62 (bs, 1H), 1.02 (bs, 1H), 0.36 (bs, 1H). TOF

ES+ MS: (M + H) 501.2, (M + Na) 523.2.

4-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(2-(pyridin-4-yl)ethyl)-

piperazine-1-carboxamide (CCG-212390). t-Butyl 4-((2-(pyridin-4-

yl)ethyl)carbamoyl)piperazine-1-carboxylate (233 mg, 0.70 mmol) was dissolved in

anhydrous 1,4-dioxane (1 mL), and 4 M HCl in 1,4-dioxane (1.0 mL, 4.18 mmol) was

added. The reaction was allowed to stir for 2 h, after which time the solvent was removed

and the residue was solidified via sonication in Et2O. The solid was washed with Et2O

then thoroughly dried under high vacuum to give the dihydrochloride salt as a tan


4-((2-(pyridin-4-yl)ethyl)carbamoyl)piperazine dihydrochloride (214 mg, 0.70

mmol), 1-(4-chlorobenzyl)-1H-indole-2-carboxylic acid (200 mg, 0.70 mmol), TEA

(0.388 mL, 2.79 mmol), EDCHCl (147 mg, 0.766 mmol), and HOBT (117 mg, 0.766

mmol) were dissolved in DCM (10 mL) and stirred for 16 h at RT. After this time, the

reaction was diluted with EtOAc and washed with H2O (3X), 10% Na2CO3 (3X), and

brine (1X), then dried (MgSO4) and concentrated. The resulting residue was purified via

silica flash chromatography (25 g silica, 1% to 20% methanolic ammonia:EtOAc) to

215

provide a white solid. Yield: 224 mg, 0.446 mmol, 64%. 1H NMR (400 MHz,

Chloroform-d) δ 8.47 (d, J = 5.4 Hz, 2H), 7.64 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 8.4 Hz,

1H), 7.31 – 7.26 (m, 1H), 7.17 (dd, J = 12.9, 7.8 Hz, 3H), 7.11 (d, J = 5.4 Hz, 2H), 6.99

(d, J = 8.2 Hz, 2H), 6.63 (s, 1H), 5.47 (s, 2H), 4.72 (t, J = 5.4 Hz, 1H), 3.60 (s, 4H), 3.49

(q, J = 6.6 Hz, 2H), 3.16 (bs, 4H), 2.82 (t, J = 6.9 Hz, 2H). TOF ES+ MS: (M + H) 502.2,

(M + Na) 524.2. HPLC tR = 5.40 min, > 95%.

1-(2-(4-Chlorobenzoyl)benzoyl)-N-(2-(pyridin-4-yl)ethyl)piperidine-4-

carboxamide (CCG-212391). 2-(4-chlorobenzoyl)benzoic acid (366 mg, 1.404 mmol)

was added to DCM (20 mL), followed by amine dihydrochloride 36 (473 mg, 1.544

mmol), TEA (978 µL, 7.02 mmol), EDCHCl (296 mg, 1.544 mmol), and HOBT (237

mg, 1.544 mmol.) The reaction was allowed to stir at RT for 20 h, at which time the

solvent was removed in vacuo. The residue was taken up in EtOAc and washed with H2O

(1X), 10% aq. Na2CO3 (3X), and brine (1X), then dried (MgSO4) and concentrated. The

residue was then purified by silica flash chromatography (25 g silica, 1% to 20% 7 M

methanolic ammonia:EtOAc.) 1H NMR (400 MHz, Chloroform-d) δ 8.47 (d, J = 5.5 Hz,

2H), 7.72 (d, J = 8.4 Hz, 2H), 7.61 – 7.53 (m, 1H), 7.52 – 7.24 (m, 5H), 7.10 (d, J = 5.4

Hz, 2H), 5.98 (t, J = 5.5 Hz, 1H), 4.47 (d, J = 12.9 Hz, 1H), 3.62 (d, J = 13.5 Hz, 1H),

3.51 (q, J = 6.6 Hz, 2H), 3.02 (t, J = 11.6 Hz, 1H), 2.81 (t, J = 6.9 Hz, 2H), 2.71 (t, J =

11.9 Hz, 1H), 2.28 (ddd, J = 14.9, 11.1, 3.9 Hz, 1H), 1.91 – 1.52 (m, 4H). TOF ES+ MS:

(M + H) 476.2, (M + Na) 498.2. HPLC tR = 4.83 min, > 95%.

216

1-(2-(4-chlorophenoxy)benzoyl)-N-(2-(pyridin-4-yl)ethyl)piperidine-4-

carboxamide (CCG-212392). Methyl 2-(4-chlorophenoxy)benzoate 41 was dissolved in

EtOH (5 mL), followed by the addition of 10% aq. NaOH (2 mL). The reaction was

allowed to stir for 14 h, after which time the solvent was stripped off in vacuo and the

residue re-dissolved in H2O. This aqueous solution was then cooled to 0 °C and acidified

to pH<1 with conc. HCl, and the resulting precipitate was collected over a filter, then

dried via aspirator and high vacuum to provide the desired carboxylic acid as a white

powder. No further purification necessary. Yield: 92 mg, 0.37 mmol, 97%.

All of the previously prepared carboxylic acid (92 mg, 0.37 mmol) was added to

DCM (5 mL), followed by amine dihydrochloride 36 (136 mg, 0.44 mmol), TEA (310

µL, 2.22 mmol), EDCHCl (85 mg, 0.44 mmol), and HOBT (68 mg, 0.44 mmol). The

solution was allowed to stir at RT for 18 h, after which time the reaction was

concentrated in vacuo and the resulting residue was taken up in EtOAc and washed with

H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X), then dried (MgSO4) and concentrated.

The resulting residue was purified by silica flash chromatography (10 g silica, 1% to 20%

methanolic ammonia:EtOAc) to give the desired compound as a thick oil. 1H NMR (500

MHz, Chloroform-d) δ 8.43 (d, J = 4.7 Hz, 2H), 7.28 (dt, J = 29.2, 7.9 Hz, 4H), 7.14 (t, J

= 7.4 Hz, 1H), 7.06 (d, J = 4.8 Hz, 2H), 6.97 – 6.76 (m, 3H), 6.15 (d, J = 47.8 Hz, 1H),

4.57 (d, J = 12.6 Hz, 1H), 3.63 (bs, 1H), 3.48 (q, J = 6.5 Hz, 2H), 3.04 – 2.88 (m, 1H),

2.77 (t, J = 6.9 Hz, 2H), 2.75 – 2.62 (m, 1H), 2.24 (t, J = 11.0 Hz, 1H), 1.81 – 1.73 (m,

1H), 1.72 – 1.61 (m, 2H), 1.61 – 1.51 (m, 1H). TOF ES+ MS: (M + H) 464.1, (M + Na)

486.1. HPLC tR = 4.79 min, > 90%.

217

1-(2-((4-Chlorophenyl)(methyl)amino)benzoyl)-N-(2-(pyridin-4-yl)ethyl)-

piperidine-4-carboxamide (CCG-212393). Methyl 2-((4-

chlorophenyl)(methyl)amino)benzoate 45 (120 mg, 0.44 mmol) was dissolved in EtOH (8

mL), followed by the addition of 10% aq. NaOH (3 mL). The reaction was allowed to stir

at RT for 6 h, after which time the solvent was removed and the residue was taken up in

H2O, cooled to 0 °C, and acidified to pH<1. The resulting white precipitate was collected

over a filter, dried under high vacuum, and used without further purification. Yield: 108

mg, 0.41 mmol, 95%. 1H NMR (400 MHz, Chloroform-d) δ 9.44 (s, 1H), 7.96 (dd, J =

8.0, 1.5 Hz, 1H), 7.35 – 7.24 (m, 3H), 7.17 (t, J = 9.0 Hz, 3H), 6.79 – 6.72 (m, 1H), 3.89

(s, 3H).

2-((4-chlorophenyl)(methyl)amino)benzoic acid (85 mg, 0.33 mmol), amine

dihydrochloride 36 (119 mg, 0.39 mmol), TEA (270 µL, 1.95 mmol), EDCHCl (75 mg,

0.39 mmol), and HOBT (60 mg, 0.39 mmol) were added to DCM (5 mL) sequentially,

and the solution was allowed to stir at RT for 20 h. After this time, the solvent was

removed in vacuo and the residue was dissolved in EtOAc and washed with H2O (3X),

10% aq. Na2CO3 (3X), and brine (1X), then dried (MgSO4) and concentrated. The residue

was then purified via silica flash chromatography (10 g silica, 1% to 20% methanolic

ammonia:EtOAc) to provide the desired compound as a thick colorless oil. Yield: 90 mg,

0.19 mmol, 58%. 1H NMR (400 MHz, Chloroform-d, mixture of rotamers) δ 8.48 (m,

2H), 7.39 (t, J = 8.1 Hz, 1H), 7.34 – 7.19 (m, 3H), 7.09 (d, J = 7.9 Hz, 4H), 6.63 (t, J =

9.6 Hz, 2H), 5.75 (t, J = 5.4 Hz, 0.5H), 5.41 (t, J = 5.4 Hz, 0.5H), 4.64 – 4.44 (m, 1H),

3.61 – 3.39 (m, 3H), 3.22 (s, 1.5H), 3.17 (s, 1.5H), 2.93 – 2.84 (m, 0.5H), 2.83 – 2.74 (m,

2H), 2.74 – 2.63 (m, 1H), 2.53 – 2.44 (m, 0.5H), 2.22 – 2.09 (m, 1H), 1.82 (bd, J = 13.1

218

Hz, 0.5H), 1.78 – 1.51 (m, 3.5H), 1.41 (bd, J = 13.2 Hz, 0.5H), 1.30 (qd, J = 12.9, 4.2 Hz,

0.5H). TOF ES+ MS: (M + H) 477.2, 499.1. HPLC tR = 5.13 min, > 90% purity.

(3aS,7aR)-5-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-2-(2-(pyridin-4-

yl)ethyl)octahydro-1H-pyrrolo[3,4-c]pyridin-1-one (CCG-212394). 5-(1-(4-

chlorobenzyl)-1H-indole-2-carbonyl)octahydro-1H-pyrrolo[3,4-c]pyridin-1-one 91 (35

mg, 0.09 mmol) was dissolved in anhydrous DMF (5 mL). A 60% oil suspension of NaH

(17 mg, 0.43 mmol) was added at RT under N2 and stirred for 1 h. 4-(2-

Bromoethyl)pyridine (19 mg, 0.10 mmol) was added and stirring continued for 22 h

under the same conditions. After this time, the solution was diluted with EtOAc and

washed with H2O (1X), 10% aq. Na2CO3 (2X), and brine (1X), dried with MgSO4, and

concentrated in vacuo. Purification was done via silica flash chromatography (10 g silica,

1 to 20% methanolic ammonia:EtOAc.) The residue was dissolved in DCM, and rapid

solvent removal afforded the title compound as a white powder. Yield: 36 mg, 0.07


7.9 Hz, 1H), 7.29 – 7.23 (m, 2H), 7.23 – 7.17 (m, 3H), 7.17 – 7.11 (m, 3H), 7.05 (d, J =

8.0 Hz, 2H), 5.45 (d, J = 7.0 Hz, 2H), 4.43 (d, J = 13.7 Hz, 1H), 4.10 (d, J = 3.8 Hz, 1H),

3.66 – 3.49 (m, 2H), 3.41 – 3.34 (m, 1H), 3.32 – 3.25 (m, 1H), 2.92 – 2.77 (m, 4H), 2.42

(bs, 2H), 2.10 (bs, 1H), 1.75 (bs, 1H), 1.31 (bs, 1H). TOF ES+ MS: (M + H) 513.2, (M +

Na) 535.2. HPLC tR = 5.53 min, > 95% purity.

1-(2-((4-Chlorobenzyl)amino)benzoyl)-N-(2-(pyridin-4-yl)ethyl)piperidine-4-

carboxamide (CCG-222660). Methyl 2-((4-chlorobenzyl)amino)benzoate 47a (80 mg,

219

0.29 mmol) was dissolved in MeOH (8 mL) and 10% aq. NaOH (4 mL) was added and

the reaction was stirred at RT for 24 h. At this time, the solvent was removed in vacuo

and the residue was dissolved in H2O and acidified with conc. HCl to pH<1 at 0 °C. The

resulting precipitate was collected over a filter and dried via aspirator then high vacuum

to afford the desired carboxylic acid as a white powder. No further purification necessary.

Yield: 75 mg, 0.29 mmol, 99%.

2-((4-chlorobenzyl)amino)benzoic acid (75 mg, 0.29 mmol) was dissolved in

DCM (12 mL) along with TEA (325 µL, 2.32 mmol), EDCHCl (67 mg, 0.35 mmol),

HOBT (53 mg, 0.35 mmol), and N-(2-(pyridin-4-yl)ethyl)piperidine-4-carboxamide

dihydrochloride (133 mg, 0.44 mmol). The solution was stirred at RT for 20 h, after

which time the DCM was stripped off in vacuo and the resulting residue was taken up in


(MgSO4) and concentrated. The residue was then purified via silica flash chromatography

(10 g silica, 1% to 30% methanolic ammonia:EtOAc) to give the desired compound.

Yield: 63 mg, 0.132 mmol, 46%. 1H NMR (500 MHz, Chloroform-d) δ 8.60 – 8.51 (m,

2H), 7.30 – 7.25 (m, 4H), 7.22 – 7.15 (m, 1H), 7.14 – 7.10 (m, 2H), 7.08 (dd, J = 7.6, 1.6

Hz, 1H), 6.67 (t, J = 7.4 Hz, 1H), 6.58 (d, J = 8.4 Hz, 1H), 5.58 (t, J = 5.2 Hz, 1H), 5.53

(t, J = 5.7 Hz, 1H), 4.33 (m, 4H), 3.57 (q, J = 6.7 Hz, 2H), 2.95 (t, J = 12.8 Hz, 2H), 2.85

(t, J = 6.9 Hz, 2H), 2.30 (tt, J = 11.1, 3.8 Hz, 1H), 1.82 (m, 2H), 1.74 (dt, J = 15.9, 7.9

Hz, 2H). TOF ES+ MS: (M + H) 477.2, (M + Na) 499.2. HPLC tR = 5.33 min, > 90%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(2-(pyridin-4-yl)ethyl)azepane-4-

carboxamide (CCG-222661). The ethyl ester 132 (160 mg, 0.37 mmol) was dissolved in

220

EtOH (6 mL) and 10% aq. NaOH (4 mL) was added and the reaction was stirred at RT

for 24 h. At this time, the solvent was removed in vacuo and the white residue was

dissolved in H2O and acidified with conc. HCl to pH<1 at 0 °C. The resulting precipitate

was collected over a filter and dried via aspirator then high vacuum to afford the desired

carboxylic acid as a white powder. No further purification necessary. Yield: 144 mg, 0.35

mmol, 96%.

The carboxylic acid (60 mg, 0.15 mmol), amine 27 (21 µL, 0.18 mmol),


mmol) were dissolved in DCM (10 mL) and stirred at RT for 29 h, at which time the




1% to 20% methanolic ammonia:EtOAc) to give a colorless oil. Yield: 40 mg, 0.76

mmol, 52%. HPLC tR = 5.53 min, > 95%. 1H NMR (500 MHz, Chloroform-d) δ 8.51 (bs,

2H), 7.65 (d, J = 7.6 Hz, 1H), 7.39 – 7.32 (m, 1H), 7.28 (s, 1H), 7.16 (d, J = 7.4 Hz, 3H),

7.12 (bs, 2H), 7.02 (d, J = 8.0 Hz, 2H), 6.65 (d, J = 6.3 Hz, 1H), 5.48 (d, J = 9.4 Hz, 2H),

3.77 – 3.64 (m, 2H), 3.60 – 3.36 (m, 4H), 3.31 – 3.15 (m, 1H), 2.86 – 2.77 (m, 2H), 2.08

– 1.97 (m, 2H), 1.91 – 1.69 (m, 3H), 1.56 – 1.48 (m, 1H), 1.43 – 1.34 (m, 1H). TOF ES+

MS: (M + H) 515.2, (M + Na) 537.2. HPLC tR = 5.52 min, > 95%.

1-(2-(4-Chlorobenzyl)benzoyl)-N-(2-(pyridin-4-yl)ethyl)piperidine-4-carboxamide

(CCG-222821). 2-(4-chlorobenzyl)benzoic acid (40 mg, 0.16 mmol), N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide dihydrochloride (55 mg, 0.18 mmol), TEA (225 µL,

221


dissolved in DCM (10 mL) and stirred at RT for 24 h. The DCM was then removed in

vacuo and the residue was taken up in EtOAc, then washed with H2O (3X), 10% aq.

Na2CO3 (3X), and brine (1X), then dried (MgSO4) and concentrated. Purification via

silica flash chromatography (4 g silica, 1% to 20% methanolic ammonia:EtOAc)

provided the desired compound as white microcrystals upon concentration. 1H NMR (500

MHz, Chloroform-d, 3:2 mixture of rotamers) δ 8.51 (bs, 2H), 7.33 (t, J = 7.3 Hz, 1H),

7.27 – 7.18 (m, 4H), 7.17 – 7.05 (m, 5H), 5.61 – 5.42 (m, 1H), 4.73 – 4.59 (m, 1H), 4.18

– 4.03 (m, 1H), 3.93 – 3.80 (m, 1H), 3.53 (q, J = 6.5 Hz, 2H), 3.37 – 3.15 (m, 1H), 2.86 –

2.81 (m, 2H), 2.50 (td, J = 12.7, 3.1 Hz, 1H), 2.23 – 1.99 (m, 2H), 1.90 – 1.75 (m, 2H),

1.73 – 1.49 (m, 2H). TOF ES+ MS: (M + H) 462.2, (M + Na) 484.2. HPLC tR = 4.90

min, > 90%.

1-(1-(4-Chlorobenzyl)-2-oxo-1,2-dihydroquinolin-4-yl)-N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (CCG-222822). Ethyl 1-(1-(4-chlorobenzyl)-2-oxo-

1,2-dihydroquinolin-4-yl)piperidine-4-carboxylate (75 mg, 0.18 mmol) was dissolved

into a mixture of EtOH (4 mL), THF (4 mL), and 10% aq. NaOH (2 mL) and stirred at

RT for 18 h. After this time, the reaction was concentrated and the residue dissolved in

H2O, chilled to 0 °C, and acidified to pH<1 with conc. HCl. The resulting precipitate was

collected over a filter and washed with cold 1M HCl, air dried via aspirator, then dried

under high vacuum to give 1-(1-(4-chlorobenzyl)-2-oxo-1,2-dihydroquinolin-4-

yl)piperidine-4-carboxylic acid as a white solid. No further purification necessary. Yield:

67 mg, 0.17 mmol, 95%.

222

1-(1-(4-chlorobenzyl)-2-oxo-1,2-dihydroquinolin-4-yl)piperidine-4-carboxylic

acid (48 mg, 0.12 mmol), 4-(2-aminoethyl)pyridine (17 µL, 0.15 mmol), TEA (51 µL,


dissolved in DCM (5 mL) and stirred at RT for 20 h. DCM was then removed in vacuo

and the residue taken up in EtOAc and washed with H2O (3X), 10% aq. Na2CO3 (3X),

and brine (1X), then dried (MgSO4) and concentrated. The residue was purified by silica

flash chromatography (10 g silica, 1% to 20% methanolic ammonia:EtOAc). Yield: 45

mg, 0.05 mmol, 45%. 1H NMR (500 MHz, Chloroform-d) δ 8.54 (s, 2H), 7.78 (d, J = 8.1

Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.21 – 7.00 (m, 6H), 6.22 (s,

1H), 5.81 (t, J = 5.4 Hz, 1H), 5.46 (s, 2H), 3.58 (dt, J = 16.9, 8.7 Hz, 4H), 2.87 (t, J = 6.9

Hz, 2H), 2.74 (t, J = 11.6 Hz, 2H), 2.28 (tt, J = 11.4, 3.7 Hz, 1H), 2.05 (qd, J = 12.5,

12.0, 3.5 Hz, 2H), 1.94 (d, J = 11.2 Hz, 2H). TOF ES+ MS: (M + H) 501.2, (M + Na)

523.2. HPLC tR = 5.25 min, >95% purity.

1-(1-(4-Chlorophenyl)-2-oxo-1,2-dihydroquinolin-4-yl)-N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (CCG-222823). Ethyl 1-(1-(4-chlorophenyl)-2-oxo-

1,2-dihydroquinolin-4-yl)piperidine-4-carboxylate 58 (150 mg, 0.37 mmol) was

dissolved in THF (10 mL) followed by the addition of 10% aq. NaOH (5 mL). This

biphasic reaction was stirred at RT for 20 h. The solvent was then stripped off in vacuo

and the resulting residue was dissolved in H2O, chilled to 0°C, then acidified to pH < 1.

The resulting precipitate was collected over a filter, air dried, then high vacuum dried to

provide 1-(1-(4-chlorophenyl)-2-oxo-1,2-dihydroquinolin-4-yl)piperidine-4-carboxylic

223

acid as a white powder. No further purification necessary. Yield: 130 mg, 0.33 mmol,

91%.

1-(1-(4-Chlorophenyl)-2-oxo-1,2-dihydroquinolin-4-yl)piperidine-4-carboxylic

acid (66 mg, 0.17 mmol), 4-(2-aminoethyl)pyridine (25 µL, 0.21 mmol), TEA (72 µL,

0.52 mmol), ED C (40 mg, 0.21 mmol), and HOBT (32 mg, 0.21 mmol) were dissolved

in DCM (5 mL) and stirred at RT for 19 h. The reaction was then diluted with EtOAc and

washed with H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X), then dried (MgSO4) and

concentrated. The residue was purified via silica flash chromatography (10 g silica, 1 to

17% methanolic ammonia/EtOAc) to give the title compound as an off-white powder.


Hz, 2H), 7.79 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.32 (t, J = 7.5 Hz, 1H), 7.25

– 7.18 (m, 3H), 7.15 (d, J = 4.9 Hz, 2H), 6.66 (d, J = 8.4 Hz, 1H), 6.20 (s, 1H), 5.78 (t, J

= 6.0 Hz, 1H), 5.30 (s, 1H), 3.58 (q, J = 9.6, 6.8 Hz, 4H), 2.85 (t, J = 6.8 Hz, 2H), 2.77 (t,

J = 11.9 Hz, 2H), 2.34 – 2.21 (m, 2H), 2.07 (q, J = 13.3 Hz, 2H), 1.95 (d, J = 13.5 Hz,

2H). TOF ES+ MS: (M + H) 487.2, (M + Na) 509.2. HPLC tR = 5.09 min, >95% purity.

1-(9H-Carbazole-1-carbonyl)-N-(2-(pyridin-4-yl)ethyl)piperidine-4-carboxamide

(CCG-222824). 9H-carbazole-1-carboxylic acid was prepared according to Journal of

Organic Chemistry, Vol. 53, No. 4, 1988, 794-799.

9H-Carbazole-1-carboxylic acid (120 mg, 0.57 mmol), N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide dihydrochloride (190 mg, 0.63 mmol), TEA (475 µL,

3.41 mmol), EDCHCl (120 mg, 0.63 mmol) and HOBT (96 mg, 0.63 mmol) were

dissolved in DCM (10 mL) and stirred at RT for 14 h. At this time, the reaction was

224

diluted with EtOAc and washed with H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X),

then dried (MgSO4) and concentrated. The residue was purified via silica flash

chromatography (10 g silica, 10% to 30% methanolic ammonia/EtOAc) to give a tan

powder. Yield: 170 mg, 0.40 mmol, 70%. 1H NMR (500 MHz, Chloroform-d) δ 9.13 (s,

1H), 8.53 (d, J = 5.6 Hz, 2H), 8.14 (d, J = 7.7 Hz, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.50 –

7.42 (m, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.26 – 7.23 (m, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.11

(d, J = 5.3 Hz, 2H), 5.59 (t, J = 5.5 Hz, 1H), 4.46 (s, 2H), 3.56 (q, J = 6.6 Hz, 2H), 3.04

(t, J = 12.3 Hz, 2H), 2.84 (t, J = 6.9 Hz, 2H), 2.32 (ddd, J = 15.0, 10.9, 4.1 Hz, 1H), 1.91

– 1.72 (m, 4H). TOF ES+ MS: (M + H) 427.2, (M + Na) 449.2. HPLC tR = 4.80 min,

>95% purity.

1-(6-Chloro-9H-carbazole-1-carbonyl)-N-(2-(pyridin-4-yl)ethyl)piperidine-4-

carboxamide (CCG-222825). Methyl 6-chloro-9H-carbazole-1-carboxylate (54 mg, 0.21

mmol) was dissolved in EtOH (4 mL) and THF (5 mL), then 10% aq. NaOH (2 mL) was

added. The reaction was allowed to stir at RT for 22 h, after which time the solvent was

stripped off in vacuo. The residue was taken up in H2O and acidified to pH<1 with conc.

HCl, and the resulting white precipitate was collected over a filter, air dried, then high

vacuum dried to give 6-chloro-9H-carbazole-1-carboxylic acid as a white powder with no

further purification. Yield: 51 mg, 0.21 mmol, 100%.

6-Chloro-9H-carbazole-1-carboxylic acid (51 mg, 0.21 mmol) was added to DCM

(5 mL), followed by N-(2-(pyridin-4-yl)ethyl)piperidine-4-carboxamide dihydrochloride

(76 mg, 0.25 mmol), TEA (145 µL, 1.04 mmol), EDCHCl (48 mg, 0.25 mmol) and

HOBT (38 mg, 0.25 mmol). This solution was stirred at RT for 16 h, at which time the

225

solvent was removed in vacuo and the residue taken up in EtOAc and washed with H2O

(3X), 10% aq. Na2CO3 (3X), and brined (1X). The extract was dried (MgSO4) and


20% methanolic ammonia: EtOAc). Yield: 48 mg, 0.10 mmol, 50%. 1H NMR (500 MHz,

Chloroform-d) δ 8.53 (d, J = 4.7 Hz, 2H), 8.10 – 8.06 (m, 1H), 8.04 – 7.98 (m, 1H), 7.48

– 7.36 (m, 3H), 7.22 (ddd, J = 7.6, 5.9, 1.0 Hz, 1H), 7.13 (d, J = 5.1 Hz, 2H), 5.61 – 5.52

(m, 1H), 4.45 (bs, 2H), 3.57 (q, J = 6.6 Hz, 2H), 3.06 (s, 2H), 2.85 (t, J = 6.9 Hz, 2H),

2.34 (tt, J = 10.9, 4.6 Hz, 1H), 1.91 – 1.69 (m, 4H). TOF ES+ MS: (M + H) 461.2, (M +

Na) 483.2. HPLC tR = 5.23 min, > 90% purity.

Exo-(1R,5S,6r)-3-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)-N-(2-(pyridin-4-

yl)ethyl)-3-azabicyclo[3.1.0]hexane-6-carboxamide (CCG-222980). The ethyl ester 113

(206 mg, 0.49 mmol) was dissolved in anhydrous THF (10 mL), followed by the addition

of KOTMS (66 mg, 0.51 mmol). The reaction stirred at RT for 48 h, solvent was

removed in vacuo, the residue was dried under high vacuum overnight, leaving the

potassium salt residue that was taken directly into the next step. Yield: 213 mg, 0.49

mmol, 100%.

The potassium salt (211 mg, 0.49 mmol), amine 27 (64 µL, 0.54 mmol), HATU

(204 mg, 0.54 mmol), and DIPEA (170 µL, 0.97 mmol) were dissolved in DCM (10 mL)

and stirred at RT for 24 h, at which time the DCM was removed in vacuo and the residue

was diluted in EtOAc and washed with H2O (2X), 10% aq. Na2CO3 (3X), and brine (1X).

The organic extract was dried (MgSO4) and concentrated, and the residue purified via

silica flash chromatography (25 g silica, 1% to 20% methanolic ammonia:EtOAc) to give

226

an off-white powder. Yield: 134 mg, 0.27 mmol, 55%. 1H NMR (500 MHz, Chloroform-

d) δ 8.44 (d, J = 5.0 Hz, 2H), 7.61 (d, J = 7.9 Hz, 1H), 7.33 – 7.24 (m, 2H), 7.17 – 7.10

(m, 3H), 7.06 (d, J = 5.0 Hz, 2H), 6.88 (d, J = 8.1 Hz, 2H), 6.66 (s, 1H), 6.07 (t, J = 5.9

Hz, 1H), 5.55 – 5.34 (m, 2H), 4.02 (d, J = 12.3 Hz, 1H), 3.72 – 3.58 (m, 2H), 3.53 – 3.32

(m, 4H), 2.75 (t, J = 7.0 Hz, 2H), 1.99 (d, J = 9.4 Hz, 2H). TOF ES+ MS: (M + H) 499.2,

(M + Na) 521.2. HPLC tR = 5.36 min, > 95%.

1-(2-(Phenylamino)benzoyl)-N-(2-(pyridin-4-yl)ethyl)piperidine-4-carboxamide

(CCG-222981). Methyl 2-(phenylamino)benzoate (74 mg, 0.33 mmol) was dissolved in

10% aq. NaOH (1 mL), THF (5 mL), and EtOH (2 mL), and stirred at RT for 5 h. At this

time, the solvent was stripped off in vacuo and the residue was dissolved in H2O and

acidified with conc. HCl to pH < 1. Material was collected over a filter and air-dried then

dried under high vacuum to give 2-(phenylamino)benzoic acid as a white powder. No

further purification necessary. Yield: 62 mg, 0.29 mmol, 89%.

2-(Phenylamino)benzoic acid (56 mg, 0.26 mmol), N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide dihydrochloride (97 mg, 0.32 mmol), EDCHCl (60

mg, 0.32 mmol), HOBT (48 mg, 0.32 mmol), and TEA (220 µL, 1.58 mmol) were

dissolved in DCM (10 mL) and stirred at RT for 17 h. After this time, the DCM was

removed in vacuo. The residue was taken up in EtOAc and washed with H2O (3X), 10%

aq. Na2CO3 (3X), and brine (1X), and dried (MgSO4) and concentrated. The residue was

purified via silica flash chromatography (10 g silica, 1% to 20% methanolic NH3/EtOAc)

to give the desired compound as a colorless oil. Yield: 53 mg, 0.12 mmol, 47%. 1H NMR

(500 MHz, Chloroform-d) δ 8.47 (d, J = 5.0 Hz, 2H), 7.37 (d, J = 8.3 Hz, 1H), 7.28 –

227

7.21 (m, 3H), 7.15 (d, J = 7.6 Hz, 1H), 7.10 – 7.02 (m, 4H), 6.93 (t, J = 7.8 Hz, 1H), 6.86

(t, J = 7.4 Hz, 1H), 5.78 (s, 1H), 4.26 (s, 2H), 3.50 (q, J = 6.5 Hz, 2H), 2.92 (bs, 2H), 2.80

(t, J = 6.9 Hz, 2H), 2.28 (ddd, J = 14.7, 11.0, 3.4 Hz, 1H), 1.77 (bs, 2H), 1.70 – 1.57 (m,

2H). TOF ES+ MS: (M + H) 429.2, (M + Na) 451.2. HPLC tR = 5.05 min, > 95%.

1-(2-(Benzylamino)benzoyl)-N-(2-(pyridin-4-yl)ethyl)piperidine-4-carboxamide

(CCG-222982). Methyl 2-(benzylamino)benzoate (50 mg, 0.21 mmol) was dissolved in

EtOH (2 mL) and THF (5 mL), then 10% aq. NaOH (2 mL) was added and the reaction

was stirred at RT for 15 h. At this time, the solvent was removed in vacuo and the residue

was dissolved in H2O and acidified with conc. HCl to pH<1. The resulting precipitate

was collected over a filter and dried via aspirator then high vacuum to afford the desired

2-(benzylamino)benzoic acid as a white powder. No further purification necessary. Yield:

41 mg, 0.18 mmol, 88%.

Ethyl 2-(benzylamino)benzoate (30 mg, 0.13 mmol) was dissolved in DCM (5

mL), followed by N-(2-(pyridin-4-yl)ethyl)piperidine-4-carboxamide dihydrochloride (45

mg, 0.15 mmol), TEA (0.11 mL, 0.79 mmol), EDCHCl (30 mg, 0.16 mmol), and HOBT

(24 mg, 0.16 mmol). The reaction was then stirred at RT for 16 h, at which time the

reaction was diluted with EtOAc and washed with H2O (2X), 10% aq. Na2CO3 (2X), and

brine (1X), then dried (MgSO4) and concentrated in vacuo. The resulting residue was

then purified by silica flash chromatography (10 g silica, 1% to 20% methanolic

ammonia:EtOAc) to provide the desired product as a colorless oil. Yield: 26 mg, 0.06


6.7 Hz, 4H), 7.26 – 7.21 (m, 1H), 7.17 (t, J = 7.8 Hz, 1H), 7.10 (d, J = 5.7 Hz, 2H), 7.07

228

– 7.02 (m, 1H), 6.63 (dd, J = 7.8, 3.8 Hz, 2H), 5.92 (t, J = 5.6 Hz, 1H), 5.44 (t, J = 5.4

Hz, 1H), 4.33 (d, J = 5.3 Hz, 2H), 4.26 (bs, 2H), 3.52 (q, J = 6.7 Hz, 2H), 2.90 (t, J = 11.7

Hz, 2H), 2.81 (t, J = 6.9 Hz, 2H), 2.28 (tt, J = 11.2, 3.5 Hz, 1H), 1.78 (d, J = 12.1 Hz,

2H), 1.69 (qd, J = 12.9, 12.4, 4.0 Hz, 2H). TOF ES+ MS: (M + H) 443.2, (M + Na)

465.2. HPLC tR = 4.92 min, > 95%.

1-(2-((4-Chlorophenethyl)amino)benzoyl)-N-(2-(pyridin-4-yl)ethyl)piperidine-4-

carboxamide (CCG-222983). 2-((4-chlorophenethyl)amino)benzoic acid (40 mg, 0.15

mmol), N-(2-(pyridin-4-yl)ethyl)piperidine-4-carboxamide dihydrochloride (53 mg, 0.17

mmol), EDCHCl (33 mg, 0.17 mmol), HOBT (26 mg, 0.17 mmol), and TEA (0.12 mL,

0.87 mmol) were dissolved in DCM (10 mL) and stirred at RT for 24 h, at which time the




1% to 20% methanolic ammonia:EtOAc) to give a colorless oil. Yield: 41 mg, 0.083

mmol, 57%. HPLC tR = 5.55 min, > 95%. 1H NMR (500 MHz, Chloroform-d) δ 8.52 (d,

J = 5.2 Hz, 2H), 7.30 – 7.21 (m, 3H), 7.16 (d, J = 8.2 Hz, 2H), 7.12 (d, J = 5.2 Hz, 2H),


5.6 Hz, 1H), 4.97 (bs, 1H), 4.21 (bs, 2H), 3.56 (q, J = 6.5 Hz, 2H), 3.34 (t, J = 7.2 Hz,

2H), 2.89 (t, J = 7.2 Hz, 2H), 2.83 (q, J = 12.6, 9.7 Hz, 4H), 2.24 (tt, J = 11.2, 3.7 Hz,

1H), 1.77 – 1.62 (m, 7H). TOF ES+ MS: (M + H) 491.2, (M + Na) 513.2. HPLC tR =

5.44 min, >95% purity.

229

(1R,3s,5S)-8-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(2-(pyridin-4-

yl)ethyl)-8-azabicyclo[3.2.1]octane-3-carboxamide (CCG-224000). The Boc-protected

right-hand half 115 (100 mg, 0.35 mmol) was dissolved into 4M HCl (5 mL) and stirred

for 15 min at RT. After this time, Et2O (15 mL) was added to precipitate the material and

the reaction was sonicated for 15 min. The resulting residue did not solidify, so the liquid

phase was decanted off and the residue was dried under aspirator vacuum, then high

vacuum, for 48 h. The resulting solid aminde dihydrochloride 116 was then taken directly

into the subsequent reaction. Yield: 115 mg, 0.35 mmol, 99%.

The indole carboxylic acid 4a (52 mg, 0.18 mmol), amine dihydrochloride 116

(60 mg, 0.18 mmol), EDCHCl (45 mg, 0.24 mmol), HOBT (36 mg, 0.24 mmol), and

TEA (252 µL, 0.24 mmol) were dissolved in DCM (3 mL) and stirred at RT for 20 h, at

which time the DCM was removed in vacuo and the residue was diluted in EtOAc and

washed with H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X). The organic extract was

dried (MgSO4) and concentrated, and the residue purified via silica flash chromatography

(10 g silica, 1% to 20% methanolic ammonia:EtOAc) to give an off-white powder. Yield:

59 mg, 0.11 mmol, 61%. 1H NMR (500 MHz, Chloroform-d) δ 8.44 (d, J = 4.8 Hz, 2H),

7.63 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.26 (t, J = 7.7 Hz, 1H), 7.19 – 7.12

(m, 3H), 7.07 (d, J = 5.0 Hz, 2H), 6.98 (d, J = 8.1 Hz, 2H), 6.74 (s, 1H), 6.11 (t, J = 5.7

Hz, 1H), 5.51 (d, J = 9.8 Hz, 2H), 4.82 (bs, 1H), 4.39 (bs, 1H), 3.48 (q, J = 6.6 Hz, 2H),

2.78 (t, J = 7.0 Hz, 2H), 2.60 (tt, J = 11.5, 5.2 Hz, 1H), 2.30 (bs, 2H), 1.76 (dd, J = 143.8,

60.1 Hz, 7H). TOF ES+ MS: (M + H) 527.2, (M + Na) 549.2. HPLC tR = 5.55 min,

>95%.

230

2-(1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)piperidin-4-yl)-N-(pyridin-4-

ylmethyl)acetamide (CCG-224001). The ethyl ester 96 (350 mg, 0.80 mmol) was

dissolved in EtOH (10 mL), THF (10 mL), and 10% aq. KOH (3.22 mL) was added and

the reaction was stirred at RT for 13 h. At this time, the solvent was removed in vacuo

and the thick white residue was diluted in H2O, sonicated, and acidified with conc. HCl to

pH<1 at 0 °C. The resulting precipitate was collected over a filter and dried via aspirator

then high vacuum to afford the desired carboxylic acid as a white powder. No further

purification necessary. Yield: 311 mg, 0.76 mmol, 95%.



mmol) were dissolved in DCM (5 mL) and stirred at RT for 18 h, at which time the DCM




20% methanolic ammonia:EtOAc) to give a colorless oil. Yield: 42 mg, 0.08 mmol, 54%.


7.39 (d, J = 8.3 Hz, 1H), 7.29 (t, J = 7.7 Hz, 1H), 7.20 – 7.14 (m, 3H), 7.13 (d, J = 5.3

Hz, 2H), 7.02 (d, J = 8.2 Hz, 2H), 6.59 (s, 1H), 6.34 (t, J = 5.7 Hz, 1H), 5.45 (s, 2H), 4.59

(bs, 1H), 4.39 (s, 2H), 4.06 (bs, 1H), 2.88 (bs, 1H), 2.67 (bs, 1H), 2.13 – 2.02 (m, 3H),

1.71 (bs, 1H), 1.45 (bs, 1H), 0.97 (bs, 1H), 0.32 (bs, 1H). TOF ES+ MS: (M + H) 501.2,

(M + Na) 523.2. HPLC tR = 5.49 min, > 95%.

231

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(2-(pyridin-4-yl)ethyl)azetidine-

3-carboxamide (CCG-224002). The methyl ester 125 (100 mg, 0.26 mmol) was

dissolved in EtOH (5 mL) and 10% aq. NaOH (3 mL) was added and the reaction was

stirred at RT for 24 h. At this time, the solvent was removed in vacuo and the white

residue was dissolved in H2O and acidified with conc. HCl to pH<1 at 0 °C. The resulting

precipitate was collected over a filter and dried via aspirator then high vacuum to afford

the desired carboxylic acid as a white powder. No further purification necessary. Yield:

95 mg, 0.26 mmol, 99%.



mmol) were dissolved in DCM (5 mL) and stirred at RT for 15 h, at which time the DCM




20% methanolic ammonia:EtOAc) to give a colorless oil. Yield: 42 mg, 0.09 mmol, 65%.


7.34 – 7.28 (m, 2H), 7.22 (d, J = 8.4 Hz, 2H), 7.19 – 7.15 (m, 1H), 7.13 (s, 2H), 7.03 (d, J

= 8.4 Hz, 2H), 6.83 (s, 1H), 5.73 (s, 2H), 5.67 (s, 1H), 4.62 (s, 1H), 4.31 (d, J = 71.6 Hz,

3H), 3.61 (q, J = 6.6 Hz, 2H), 3.22 (p, J = 7.4 Hz, 2H), 2.87 (t, J = 7.0 Hz, 2H). TOF ES+

MS: (M + H) 473.2, (M + Na) 495.2. HPLC tR = 5.60 min, >95% purity.

2-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(2-(pyridin-4-yl)ethyl)-2-

azaspiro[3.3]heptane-6-carboxamide (CCG-224220). In a dry round bottom flask under

232

argon at RT, naphthalene (220 mg, 1.74 mmol) was dissolved in anhydrous THF (10 mL)

containing hexane-washed sodium (100 mg, 4.35 mmol). The mixture was sonicated until

the reaction became a dark green homogenous solution and no solid material was visible.

This entire solution was then added dropwise to tosyl amide 123 (41 mg, 0.10 mmol),

dissolved in anhydrous THF (1 mL), which caused the reaction to turn turbid brown. This

was allowed to stir for 5 min. After this time, the reaction was diluted with EtOAc and a

few drops of MeOH and washed with H2O (1X), 10% Na2CO3 (3X), and brine (1X), then

dried (MgSO4) and concentrated. The residue of amine 124 was used directly in the

subsequent reaction.

The indole carboxylic acid 4a (29 mg, 0.10 mmol), spiro amine 124 (approx. 30

mg, 0.10 mmol), HATU (49 mg, 0.12 mmol), and TEA (42 µL, 0.30 mmol) were

dissolved in DMF (2 mL) and stirred at RT for 18 h, at which time the reaction was

diluted with EtOAc and washed with H2O (3X), 10% aq. Na2CO3 (3X), and brine (1X).

The organic extract was dried (MgSO4) and concentrated, and the residue purified via

silica flash chromatography (4 g silica, 1% to 20% methanolic ammonia:EtOAc) to give a

colorless oil. Yield: 18 mg, 0.04 mmol, 35%. 1H NMR (500 MHz, Chloroform-d) δ 8.50

(d, J = 4.5 Hz, 2H), 7.65 (d, J = 7.7 Hz, 1H), 7.28 (d, J = 16.1 Hz, 3H), 7.19 – 7.13 (m,

2H), 7.11 (d, J = 5.2 Hz, 2H), 6.99 (d, J = 7.6 Hz, 2H), 6.80 (s, 1H), 5.71 (s, 2H), 5.58

(bs, 1H), 4.28 (bd, J = 32.5 Hz, 2H), 4.06 (s, 2H), 3.52 (q, J = 6.7 Hz, 2H), 2.82 (t, J =

7.0 Hz, 2H), 2.74 (bs, 1H), 2.49 – 2.29 (m, 4H). TOF ES+ MS: (M + H) 513.2, (M + Na)

535.2. HPLC tR = 5.65 min, > 95%.

233

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